CN107522784A

CN107522784A - The anti-antibody of IL 23

Info

Publication number: CN107522784A
Application number: CN201710917166.3A
Authority: CN
Inventors: R.R.巴里特; K.卡纳达; K.M.卡特龙; R.科彭哈佛; L.E.弗里戈; E.L.雷蒙德; S.辛格; X.朱
Original assignee: Boehringer Ingelheim International GmbH
Current assignee: Boehringer Ingelheim International GmbH
Priority date: 2010-11-04
Filing date: 2011-11-02
Publication date: 2017-12-29
Anticipated expiration: 2031-11-02
Also published as: LTC2635601I2; PT2635601T; BR112013011065A2; HK1247940A1; AU2018236692C1; IL225648A; CA2816950C; ES2593754T3; LUC00132I2; TWI653242B; AP2013006820A0; SG10201604605VA; UY33703A; JP6445596B2; CY1118014T1; LUC00132I1; GEP201706733B; HUE030916T2; EA030436B1; JP2019054809A

Abstract

The present invention relates to the anti-IL 23p19 antibody of anti-IL 23p19 binding compounds, particularly humanization；And the treatment using it and diagnostic method and composition.

Description

Anti-il-23 antibodies

The application is the applying date on November 2nd, 2011, Chinese Application No. 201180064083.5, entitled The divisional application of the patent application of " anti-il-23 antibodies ".

Technical field

The present invention generally relates to diagnostic and therapeutic use anti-il-23 p 19 antibodies.More particularly, it is open It is used for the humanization anti-il-23 p 19 antibodies and application method for treating various diseases or illness.Also disclose comprising these chemical combination The pharmaceutical composition and kit of thing.

Background of invention

Higher eucaryote can play complicated response through evolving to pathogen, the response originated by innate immune response and It is then adaptive immune response.This two kinds of mechanism are not only eradicated the pathogen of infection biological body but also also produced for not together Come the permanent immunity response exposed.Lacking these reactions can make adaptive immune response increase the sensitiveness for infecting and/or changing Add, cause chronic inflammation and LADA.IL-12 for it is a kind of by the heterodimeric cell that p40 and p35 protein subunits form because Son, it is considered as the significant cell factor of innate immune response for a long time, has larger shadow to compliance immunity Ring.However, the data on the research of the biological action of this cell factor produce puzzling result.For example, although lack The arthritis (CIA) and Experimental Autoimmune encephalomyelitis (EAE) that weary p40 mouse is induced collagen are resistant, But lack the p35 susceptible disease suffered from both the above disease and even show deterioration of mouse.These problems are with generation nineteen ninety Later stage finds have the not IL-12 cytokine family newcomer IL-23 of same-action in immune response and start to be addressed.

IL-23 is made up of the subunit (p40) shared with IL-12 and unique p19 subunits.Although it is sub- single to share this p40 Member, but IL-23 and IL-12 effect is far from it.IL-12 is anti-to Th1 via promotion Th1 cell differentiations, propagation and activation Should be particularly important.By contrast, IL-23 supports the one group of CD4 determined recently⁺The development and maintenance of t helper cell, the CD4⁺T Auxiliary cell is due to that can produce IL-17 and relevant cell factor and referred to as Th17 cells.Increasing evidence shows IL-23 Chronic autoimmune inflammation is participated in, and the regulation of IL-23 activity can provide the promising therapy for autoimmune disease.

Therefore it is necessary to have the antagonist molecules for IL-23 of beneficial pharmacological property, it can be used as treating mankind's disease Disease, particularly immune and autoimmune disease therapeutic agent.

Therefore, an object of the invention is to provide anti-il-23 antagonist molecules, particularly has high tie to IL-23 Close the anti-il-23 antagonist molecules of affinity.

The another of the present invention aims at the anti-il-23 antagonist molecules for providing and having high specific to IL-23.

The another of the present invention aims at anti-il-23 of the association with high blocking activity provided to IL-23 and its acceptor Antagonist.

Another aim at of the present invention provides the anti-il-23 antagonist with strength cytoactive.

Another aim at of the present invention provides the anti-il-23 antagonist with favourable bioavilability.

Another aim at of the present invention provides the anti-il-23 antagonist with favourable biophysical properties.

Other targets of the present invention include the combination of any of the above described target.

Summary of the invention

The present invention solves to need and provide above the antibody combined with the p19 subunits of IL-23 albumen.In one aspect In, antibody of the invention is combined with high-affinity with mankind IL-23.In another aspect, antibody of the invention suppresses mice spleen Cell stimulates through IL-23 and produces IL-17.In another aspect, antibody of the invention not with the closely related family as IL-23 The IL-12 of member combines also not antagonism IL-12.

In one embodiment, the present invention provides the anti-il-23 p 19 antibodies as caused by mouse fusion knurl, such as Dan Ke Grand antibody.In one embodiment, the present invention provides total length anti-il-23 p 19 antibodies.In another embodiment, this hair Bright offer anti-IL-23 p 19 humanized antibody, such as Humanized monoclonal anti-il-23 p 19 antibodies, such as total length humanization Dan Ke Grand anti-il-23 p 19 antibodies.In an aspect, humanized antibody of the invention is combined with high-affinity with mankind IL-23. In another aspect, humanized antibody of the invention is also combined with high-affinity with stump-tailed macaque (cynomolgus) IL-23.Another In aspect, humanized antibody of the invention suppresses the STAT3 phosphorylations induced in DB cells by IL-23.In another aspect, originally The humanized antibody of invention is combined come antagonism IL-23 effect by the p19 subunits with IL-23, for example, by IL-17 and Measured by the suppression of IL-22 cell factor, the cell factor is stimulated by IL-23 and produced.In another aspect, it is of the invention Humanized antibody there is favourable pharmacokinetics (PK) overview.In another aspect, humanized antibody of the invention has Favourable biophysical properties, such as quality, stability or dissolubility, such as by the percentage institute in monomeric form antibody really It is fixed.

Other embodiments cover the DNA molecular of antibody of the coding present invention, the expression vector comprising these DNA moleculars and Host cell and the method for preparing antibody of the invention.The present invention additionally provides the antibody of the present invention especially for immune and The therapeutic use of autoimmune disease.

In one embodiment, the present invention additionally provides a kind of anti-il-23 p 19 antibodies or its antigen-binding fragment, its Include light chain CDR1 (L-CDR1) sequence SEQ ID NO:1st, 4,6,7,8,11,15,18,19,22,27 or 30；Light chain CDR2 (L- CDR2) sequence SEQ ID NO:2nd, 5,9,12,16,20,23,25,28 or 31；Light chain CDR3 (L-CDR3) sequence SEQ ID NO: 3rd, 10,13,14,17,21,24,26,29 or 32；Heavy chain CDR1 (H-CDR1) sequence SEQ ID NO:33、36、38、40、43、 45th, 48,51,54,57,60,63,66,67,68,69,77 or 80；Heavy chain CDR2 (H-CDR2) sequence SEQ ID NO:34、39、 41st, 46,49,52,55,58,61,64,70,72,73,75,78 or 81；And heavy chain CDR3 (H-CDR3) sequence SEQ ID NO: 35th, 37,42,44,47,50,53,56,59,62,65,71,74,76,79 or 82.In one embodiment, anti-IL-23 p 19 Antibody or its antigen-binding fragment include：Light chain variable district, the light chain variable district include L-CDR1 listed above, L- listed above CDR2 and L-CDR3 listed above；And weight chain variable district, the weight chain variable district include H-CDR1 listed above, H- listed above CDR2 and H-CDR3 listed above.

In one embodiment, the present invention additionally provides a kind of anti-il-23 p 19 antibodies or its antigen-binding fragment, its In the antibody or its antigen-binding fragment include respectively SEQ ID NO:1st, 2,3,33,34 and 35 L-CDR1, L-CDR2, L- CDR3, H-CDR1, H-CDR2 and H-CDR3 sequence；Or respectively SEQ ID NO:4th, 5,3,36,34 and 37 L-CDR1, L- CDR2, L-CDR3, H-CDR1, H-CDR2 and H-CDR3 sequence；Or respectively SEQ ID NO:1st, 2,3,38,39 and 35 L- CDR1, L-CDR2, L-CDR3, H-CDR1, H-CDR2 and H-CDR3 sequence；Or respectively SEQ ID NO:6th, 2,3,40,41 and 42 L-CDR1, L-CDR2, L-CDR3, H-CDR1, H-CDR2 and H-CDR3 sequence；Or respectively SEQ ID NO:7、2、3、 43rd, 41 and 44 L-CDR1, L-CDR2, L-CDR3, H-CDR1, H-CDR2 and H-CDR3 sequence；Or respectively SEQ ID NO: 8th, 9,10,45,46 and 47 L-CDR1, L-CDR2, L-CDR3, H-CDR1, H-CDR2 and H-CDR3 sequence；Respectively SEQ ID NO:8th, 9,10,48,49 and 50 L-CDR1, L-CDR2, L-CDR3, H-CDR1, H-CDR2 and H-CDR3 sequence；Or it is respectively SEQ ID NO:11st, 12,13,51,52 and 53 L-CDR1, L-CDR2, L-CDR3, H-CDR1, H-CDR2 and H-CDR3 sequence； Or respectively SEQ ID NO:7th, 2,14,54,55 and 56 L-CDR1, L-CDR2, L-CDR3, H-CDR1, H-CDR2 and H- CDR3 sequences；Or respectively SEQ ID NO:15th, 16,17,57,58 and 59 L-CDR1, L-CDR2, L-CDR3, H-CDR1, H- CDR2 and H-CDR3 sequences；Or respectively SEQ ID NO:18th, 16,17,60,61 and 62 L-CDR1, L-CDR2, L-CDR3, H-CDR1, H-CDR2 and H-CDR3 sequence；Or respectively SEQ ID NO:19；20；21；63rd, 66,67 or 68；64；And 65 L- CDR1, L-CDR2, L-CDR3, H-CDR1, H-CDR2 and H-CDR3 sequence；Or respectively SEQ ID NO:22、23、24、69、70 And 71 L-CDR1, L-CDR2, L-CDR3, H-CDR1, H-CDR2 and H-CDR3 sequence；Or respectively SEQ ID NO:22、25、 26th, 55,72 and 71 L-CDR1, L-CDR2, L-CDR3, H-CDR1, H-CDR2 and H-CDR3 sequence；Or respectively SEQ ID NO:8th, 9,10,45,73 and 74 L-CDR1, L-CDR2, L-CDR3, H-CDR1, H-CDR2 and H-CDR3 sequence；Or it is respectively SEQ ID NO:27th, 28,29,45,75 and 76 L-CDR1, L-CDR2, L-CDR3, H-CDR1, H-CDR2 and H-CDR3 sequence； Or respectively SEQ ID NO:8th, 9,10,77,78 and 79 L-CDR1, L-CDR2, L-CDR3, H-CDR1, H-CDR2 and H- CDR3 sequences；Or respectively SEQ ID NO:30th, 31,32,80,81 and 82 L-CDR1, L-CDR2, L-CDR3, H-CDR1, H- CDR2 and H-CDR3 sequences.In one embodiment, anti-il-23 p 19 antibodies or its antigen-binding fragment include：Light chain variable Area, the light chain variable district combine comprising L-CDR1, L-CDR2 and L-CDR3 listed above；And weight chain variable district, the weight chain variable Combined comprising H-CDR1, H-CDR2 and H-CDR3 listed above in area.

In one embodiment, the present invention additionally provides a kind of anti-il-23 p 19 antibodies or its antigen-binding fragment, its In the antibody or its antigen-binding fragment contain amino acid sequence SEQ ID NO:84 light chain variable district and contain amino Acid sequence SEQ ID NO:121 weight chain variable district；Or contain amino acid sequence SEQ ID NO:86 light chain variable district and contain There are amino acid sequence SEQ ID NO:123 weight chain variable district；Or contain amino acid sequence SEQ ID NO:88 light chain variable Area and contain amino acid sequence SEQ ID NO:125 weight chain variable district；Or contain amino acid sequence SEQ ID NO:90 it is light Chain variable region and contain amino acid sequence SEQ ID NO:127 weight chain variable district；Or contain amino acid sequence SEQ ID NO: 91 light chain variable district and contain amino acid sequence SEQ ID NO:128 weight chain variable district；Or contain amino acid sequence SEQ ID NO:93 light chain variable district and contain amino acid sequence SEQ ID NO:130 weight chain variable district；Or contain amino acid sequence Arrange SEQ ID NO:95 light chain variable district and contain amino acid sequence SEQ ID NO:132 weight chain variable district；Or contain ammonia Base acid sequence SEQ ID NO:97 light chain variable district and contain amino acid sequence SEQ ID NO:134 weight chain variable district；Or Contain amino acid sequence SEQ ID NO:99 light chain variable district and contain amino acid sequence SEQ ID NO:136 weight chain variable Area；Or contain amino acid sequence SEQ ID NO:101 light chain variable district and contain amino acid sequence SEQ ID NO:138 weight Chain variable region；Or contain amino acid sequence SEQ ID NO:103 light chain variable district and contain amino acid sequence SEQ ID NO: 140 weight chain variable district；Or contain amino acid sequence SEQ ID NO:105 light chain variable district and contain amino acid sequence SEQ ID NO:142 weight chain variable district；Or contain amino acid sequence SEQ ID NO:107 light chain variable district and contain amino acid sequence Arrange SEQ ID NO:144 weight chain variable district；Or contain amino acid sequence SEQ ID NO:109 light chain variable district and contain ammonia Base acid sequence SEQ ID NO:146 weight chain variable district；Or contain amino acid sequence SEQ ID NO:111 light chain variable district and Contain amino acid sequence SEQ ID NO:148 weight chain variable district；Or contain amino acid sequence SEQ ID NO:113 light chain can Become area and contain amino acid sequence SEQ ID NO:150 weight chain variable district；Or contain amino acid sequence SEQ ID NO:115 Light chain variable district and contain amino acid sequence SEQ ID NO:152 weight chain variable district；Or contain amino acid sequence SEQ ID NO:117 light chain variable district and contain amino acid sequence SEQ ID NO:154 weight chain variable district；Or contain amino acid sequence SEQ ID NO:119 light chain variable district and contain amino acid sequence SEQ ID NO:156 weight chain variable district.

In one embodiment, the present invention additionally provides a kind of anti-il-23 p 19 antibodies or its antigen-binding fragment, its In the antibody or its antigen-binding fragment include containing being selected from SEQ ID NO:158th, 160,162 and 164 amino acid sequence Light chain variable district and containing selected from SEQ ID NO:166th, the weight chain variable district of 168,170 and 172 amino acid sequence.

In one embodiment, the present invention additionally provides a kind of anti-il-23 p 19 antibodies or its antigen-binding fragment, its In the antibody or its antigen-binding fragment to IL-23 K_DLess than 40pM or less than 20pM or less than 10pM or less than 1pM.

In another embodiment, the present invention provides a kind of anti-il-23 p 19 antibodies or its antigen-binding fragment, its By SEQ ID NO:181 amino acid residue 108 to 126 and amino acid residue 137 to 151 composition epitope at mankind IL- 23p19 is combined.

In another embodiment, the present invention provides a kind of anti-il-23 p 19 antibodies or its antigen-binding fragment, its with The antibody competition combination mankind IL-23p19 of the present invention.In one embodiment, the present invention provides a kind of anti-IL-23 p 19 Antibody or its antigen-binding fragment, its with comprising containing amino acid sequence SEQ ID NO:174 light chain and contain amino acid sequence Arrange SEQ ID NO:The Humanized monoclonal anti-il-23 p 19 antibodies competitive binding mankind IL-23p19 of 176 heavy chain.One In individual embodiment, the present invention provides a kind of anti-il-23 p 19 antibodies or its antigen-binding fragment, its with comprising containing amino acid Sequence SEQ ID NO:174 light chain and contain amino acid sequence SEQ ID NO:The anti-IL- of Humanized monoclonal of 178 heavy chain 23p19 antibody competition combination mankind IL-23p19.In one embodiment, the present invention provides a kind of anti-il-23 p 19 antibodies Or its antigen-binding fragment, its with comprising containing amino acid sequence SEQ ID NO:180 light chain and contain amino acid sequence SEQ ID NO:The Humanized monoclonal anti-il-23 p 19 antibodies competitive binding mankind IL-23p19 of 176 heavy chain.In an implementation In scheme, the present invention provides a kind of anti-il-23 p 19 antibodies or its antigen-binding fragment, its with comprising containing amino acid sequence SEQ ID NO:180 light chain and contain amino acid sequence SEQ ID NO:The Humanized monoclonal anti-IL-23 p 19 of 178 heavy chain resists Body competitive binding mankind IL-23p19.

In one embodiment, anti-il-23 p 19 antibodies are humanized antibody.In one embodiment, anti-IL- 23p19 antibody is monoclonal antibody.In one embodiment, anti-il-23 p 19 antibodies are full length antibody.In an embodiment party In case, anti-il-23 p 19 antibodies are Humanized monoclonal antibodies, such as total length Humanized monoclonal antibodies.In an embodiment In, antigen-binding fragment Fab, F (ab')₂Or Single-Chain Fv Fragment of Murine.In one embodiment, antigen-binding fragment includes light Chain variable region and weight chain variable district.

In one embodiment, the present invention additionally provides a kind of anti-il-23 p 19 antibodies or its antigen-binding fragment, its In the antibody or its antigen-binding fragment include amino acid sequence SEQ ID NO:19(CDR1-L)；Amino acid sequence SEQ ID NO:20(CDR2-L)；Amino acid sequence SEQ ID NO:21(CDR3-L)；Amino acid sequence SEQ ID NO:63rd, 66,67 or 68 (CDR1-H)；Amino acid sequence SEQ ID NO:64(CDR2-H)；And amino acid sequence SEQ ID NO:65(CDR3-H).

In one embodiment, the present invention additionally provides a kind of anti-il-23 p 19 antibodies or its antigen-binding fragment, its In the antibody or its antigen-binding fragment include amino acid sequence SEQ ID NO:19(CDR1-L)；Amino acid sequence SEQ ID NO:20(CDR2-L)；Amino acid sequence SEQ ID NO:21(CDR3-L)；Amino acid sequence SEQ ID NO:66(CDR1-H)； Amino acid sequence SEQ ID NO:64(CDR2-H)；And amino acid sequence SEQ ID NO:65(CDR3-H).

In one embodiment, the present invention additionally provides a kind of anti-il-23 p 19 antibodies or its antigen-binding fragment, its In the antibody or its antigen-binding fragment include：Light chain variable district, the light chain variable district include amino acid sequence SEQ ID NO: 19 (CDR1-L), amino acid sequence SEQ ID NO:20 (CDR2-L) and amino acid sequence SEQ ID NO:21(CDR3-L)；And Weight chain variable district, the weight chain variable district include amino acid sequence SEQ ID NO:63rd, 66,67 or 68 (CDR1-H), amino acid sequence Arrange SEQ ID NO:64 (CDR2-H) and amino acid sequence SEQ ID NO:65(CDR3-H).

In one embodiment, the present invention additionally provides a kind of anti-il-23 p 19 antibodies or its antigen-binding fragment, its In the antibody or its antigen-binding fragment include：Light chain variable district, the light chain variable district include amino acid sequence SEQ ID NO: 19 (CDR1-L), amino acid sequence SEQ ID NO:20 (CDR2-L) and amino acid sequence SEQ ID NO:21(CDR3-L)；And Weight chain variable district, the weight chain variable district include amino acid sequence SEQ ID NO:66 (CDR1-H), amino acid sequence SEQ ID NO:64 (CDR2-H) and amino acid sequence SEQ ID NO:65(CDR3-H).

In one embodiment, the present invention additionally provides a kind of anti-il-23 p 19 antibodies or its antigen-binding fragment, its In the antibody or its antigen-binding fragment include：Light chain variable district, the light chain variable district include amino acid sequence SEQ ID NO: 158th, any one of 160,162 or 164；And weight chain variable district, the weight chain variable district include amino acid sequence SEQ ID NO: 166th, any one of 168,170 or 172.

In one embodiment, the present invention additionally provides a kind of anti-il-23 p 19 antibodies or its antigen-binding fragment, its In the antibody or its antigen-binding fragment contain amino acid sequence SEQ ID NO:160 light chain variable district and contain amino Acid sequence SEQ ID NO:166 weight chain variable district.

In one embodiment, the present invention additionally provides a kind of anti-il-23 p 19 antibodies or its antigen-binding fragment, its In the antibody or its antigen-binding fragment contain amino acid sequence SEQ ID NO:160 light chain variable district and contain amino Acid sequence SEQ ID NO:168 weight chain variable district.

In one embodiment, the present invention additionally provides a kind of anti-il-23 p 19 antibodies or its antigen-binding fragment, its In the antibody or its antigen-binding fragment contain amino acid sequence SEQ ID NO:158 light chain variable district and contain amino Acid sequence SEQ ID NO:166 weight chain variable district.

In one embodiment, the present invention additionally provides a kind of anti-il-23 p 19 antibodies or its antigen-binding fragment, its In the antibody or its antigen-binding fragment contain amino acid sequence SEQ ID NO:158 light chain variable district and contain amino Acid sequence SEQ ID NO:168 weight chain variable district.

In one embodiment, anti-il-23 p 19 antibodies are humanized antibody.In one embodiment, anti-IL- 23p19 antibody is monoclonal antibody.In one embodiment, anti-il-23 p 19 antibodies are full length antibody.In an embodiment party In case, anti-il-23 p 19 antibodies are Humanized monoclonal anti-il-23 p 19 antibodies, such as total length Humanized monoclonal antibodies.One In individual embodiment, antigen-binding fragment Fab, F (ab')₂Or Single-Chain Fv Fragment of Murine.In one embodiment, antigen binding Fragment includes light chain variable district and weight chain variable district.

In one embodiment, the present invention additionally provides one kind include with IgG 1, IgG2, IgG3, IgG4, IgM, The amino acid sequence SEQ ID NO of IgA or IgE heavy chain constant region connection:166 or 168 antibody.One kind includes and IgG 1 The amino acid sequence SEQ ID NO of heavy chain constant region connection:166 or 168 antibody.One kind includes constant with mankind κ or lambda light chain The amino acid sequence SEQ ID NO of area's connection:158 or 160 antibody.It is a kind of to include the ammonia being connected with human kappa light chain's constant region Base acid sequence SEQ ID NO:158 or 160 antibody.

In one embodiment, the present invention additionally provides a kind of antibody, it is included connects with the heavy chain constant region of IgG 1 The amino acid sequence SEQ ID NO connect:The 166 or 168 and amino acid sequence SEQ ID that are connected with human kappa light chain's constant region NO:158 or 160.

In one embodiment, the present invention additionally provides a kind of Humanized monoclonal anti-il-23 p 19 antibodies, it is included Containing selected from SEQ ID NO:158th, the light chain variable district of 160,162 and 164 amino acid sequence of any one and containing selected from SEQ ID NO:166th, the weight chain variable district of 168,170 and 172 amino acid sequence of any one.

In one embodiment, the present invention additionally provides a kind of Humanized monoclonal anti-il-23 p 19 antibodies, it is included Contain amino acid sequence SEQ ID NO:160 light chain variable district and contain amino acid sequence SEQ ID NO:166 heavy chain can Become area.

In one embodiment, the present invention additionally provides a kind of Humanized monoclonal anti-il-23 p 19 antibodies, it is included Contain amino acid sequence SEQ ID NO:160 light chain variable district and contain amino acid sequence SEQ ID NO:168 heavy chain can Become area.

In one embodiment, the present invention additionally provides a kind of Humanized monoclonal anti-il-23 p 19 antibodies, it is included Contain amino acid sequence SEQ ID NO:158 light chain variable district and contain amino acid sequence SEQ ID NO:166 heavy chain can Become area.

In one embodiment, the present invention additionally provides a kind of Humanized monoclonal anti-il-23 p 19 antibodies, it is included Contain amino acid sequence SEQ ID NO:158 light chain variable district and contain amino acid sequence SEQ ID NO:168 heavy chain can Become area.

In one embodiment, the present invention additionally provides a kind of Humanized monoclonal anti-il-23 p 19 antibodies, it is included Contain amino acid sequence SEQ ID NO:174 or 180 light chain and contain amino acid sequence SEQ ID NO:176 or 178 weight Chain.

In one embodiment, the present invention additionally provides a kind of Humanized monoclonal anti-il-23 p 19 antibodies, it is included Contain amino acid sequence SEQ ID NO:174 light chain and contain amino acid sequence SEQ ID NO:176 heavy chain.

In one embodiment, the present invention additionally provides a kind of Humanized monoclonal anti-il-23 p 19 antibodies, it is included Contain amino acid sequence SEQ ID NO:174 light chain and contain amino acid sequence SEQ ID NO:178 heavy chain.

In one embodiment, the present invention additionally provides a kind of Humanized monoclonal anti-il-23 p 19 antibodies, it is included Contain amino acid sequence SEQ ID NO:180 light chain and contain amino acid sequence SEQ ID NO:176 heavy chain.

In one embodiment, the present invention additionally provides a kind of Humanized monoclonal anti-il-23 p 19 antibodies, it is included Contain amino acid sequence SEQ ID NO:180 light chain and contain amino acid sequence SEQ ID NO:178 heavy chain.

In another embodiment, the present invention relates to a kind of anti-il-23 p 19 antibodies or its antigen-binding fragment, it is wrapped Contain：Humanization light-chain variable domain, the light-chain variable domain include SEQ ID NO:160 CDR, and amino acid sequence and SEQ ID NO:The consistent skeleton area of the skeleton region amino acid sequence at least 90% of 160 variable domain light-chain amino acid sequence；And humanization weight Chain variable domain, the heavy chain variable domain include SEQ ID NO:166 CDR, and amino acid sequence and SEQ ID NO:166 can The consistent skeleton area of the skeleton region amino acid sequence at least 90% of domain heavy chain amino acid sequence.In one embodiment, resist IL-23p19 antibody is Humanized monoclonal antibodies, such as total length Humanized monoclonal antibodies.

In another embodiment, the present invention relates to a kind of anti-il-23 p 19 antibodies or its antigen-binding fragment, it is wrapped Contain：Humanization light-chain variable domain, the light-chain variable domain include SEQ ID NO:160 CDR, and amino acid sequence and SEQ ID NO:The consistent skeleton area of the skeleton region amino acid sequence at least 90% of 160 variable domain light-chain amino acid sequence；And humanization weight Chain variable domain, the heavy chain variable domain include SEQ ID NO:168 CDR, and amino acid sequence and SEQ ID NO:168 can The consistent skeleton area of the skeleton region amino acid sequence at least 90% of domain heavy chain amino acid sequence.In one embodiment, resist IL-23p19 antibody is Humanized monoclonal antibodies, such as total length Humanized monoclonal antibodies.

In another embodiment, the present invention relates to a kind of anti-il-23 p 19 antibodies or its antigen-binding fragment, it is wrapped Contain：Humanization light-chain variable domain, the light-chain variable domain include SEQ ID NO:158 CDR, and amino acid sequence and SEQ ID NO:The consistent skeleton area of the skeleton region amino acid sequence at least 90% of 158 variable domain light-chain amino acid sequence；And humanization weight Chain variable domain, the heavy chain variable domain include SEQ ID NO:166 CDR, and amino acid sequence and SEQ ID NO:166 can The consistent skeleton area of the skeleton region amino acid sequence at least 90% of domain heavy chain amino acid sequence.In one embodiment, resist IL-23p19 antibody is Humanized monoclonal antibodies, such as total length Humanized monoclonal antibodies.

In another embodiment, the present invention relates to a kind of anti-il-23 p 19 antibodies or its antigen-binding fragment, it is wrapped Contain：Humanization light-chain variable domain, the light-chain variable domain include SEQ ID NO:158 CDR, and amino acid sequence and SEQ ID NO:The consistent skeleton area of the skeleton region amino acid sequence at least 90% of 158 variable domain light-chain amino acid sequence；And humanization weight Chain variable domain, the heavy chain variable domain include SEQ ID NO:168 CDR, and amino acid sequence and SEQ ID NO:168 can The consistent skeleton area of the skeleton region amino acid sequence at least 90% of domain heavy chain amino acid sequence.In one embodiment, resist IL-23p19 antibody is Humanized monoclonal antibodies, such as total length Humanized monoclonal antibodies.

In an aspect, the feature of humanization anti-il-23 p 19 antibodies of the invention can be furthermore that to mankind IL- 23 K_DLess than or equal to 1pM.In an aspect, association rate is unchanged in 50% human serum.

In an aspect, the feature of humanization anti-il-23 p 19 antibodies of the invention can be furthermore that its live body extrernal resistance Disconnected IL-23 is combined with mankind IL-23R/Fc.

In an aspect, the feature of humanization anti-il-23 p 19 antibodies of the invention can be furthermore that its not with the mankind IL-12 is combined.

In an aspect, the feature of humanization anti-il-23 p 19 antibodies of the invention can be furthermore that it suppresses mouse The IL-17 induced in splenocyte by mankind IL-23 is produced, IC₅₀Less than or equal to 20pM.

In an aspect, the feature of humanization anti-il-23 p 19 antibodies of the invention can be furthermore that it suppresses the mankind The STAT3 phosphorylations induced in DB cells by mankind IL-23, IC₅₀Less than or equal to 40pM.

In an aspect, the feature of humanization anti-il-23 p 19 antibodies of the invention can be furthermore that it without pre- The ADCC/CDC activity of survey.

In an aspect, the feature of humanization anti-il-23 p 19 antibodies of the invention can be furthermore that it to stump-tailed macaque IL-23 K_DLess than or equal to 1pM.

In an aspect, the feature of humanization anti-il-23 p 19 antibodies of the invention can be furthermore that its do not have with Mouse or rat IL-23 cross reactivity.

In an aspect, the feature of humanization anti-il-23 p 19 antibodies of the invention can be furthermore that it suppresses mouse By the mankind IL-23 IL-17 induced and IL-22 generation in ear, the suppression to two kinds of cell factors under 1mg/kg is 80% or more than 80%.

In an aspect, the feature of humanization anti-il-23 p 19 antibodies of the invention can be furthermore that as passed through differential The melting temperature that scanning calorimetry is determined is 83 DEG C.

In an aspect, the feature of humanization anti-il-23 p 19 antibodies of the invention can be furthermore that such as by ultraviolet Spectroscopy measurements and by turbidity monitor solubility be more than or equal to 100mg/ml.

In another aspect, the feature of humanization anti-il-23 p 19 antibodies of the invention can be furthermore that it with least 90% monomeric form or at least 92% monomeric form or at least 95% monomeric form is present in buffer solution.

In another aspect, the feature of humanization anti-il-23 p 19 antibodies of the invention can be furthermore that it with least 90% monomeric form or at least 92% monomeric form or at least 95% monomeric form, which are maintained in buffer solution, continues one month or four Individual month.

In an aspect, humanization anti-il-23 p 19 antibodies are Humanized monoclonal antibodies, such as total length humanization list Clonal antibody.

Other embodiments cover the DNA molecular of coding above light chain variable district, encode DNA points of above weight chain variable district The DNA molecular of son, the DNA molecular in coding above light chain area or coding above heavy chain region.

Other embodiments cover the expression vector containing above DNA molecular.In one embodiment, expression vector bag Containing the DNA molecular for being separately encoded constant heavy and/or constant light, its respectively with coding variable heavy chain and/or variable light DNA molecular connects.Other embodiments cover the host cell for carrying one or more above-mentioned expression vectors.In an embodiment In, host is mammalian cell.

Other embodiments cover a kind of above-mentioned antibody of generation or the method for its antigen-binding fragment, and it is included with above-mentioned one Or variety carrier transfection of mammalian host cell, the host cell is cultivated, and reclaim and purify the antibody or its antigen binding Fragment.

Other embodiments cover a kind of above-mentioned antibody of generation or the method for its antigen-binding fragment, and it includes being included The mammalian host cell of one or more above-mentioned carriers, and cultivate the host cell.In one embodiment, this method is another The antibody or its antigen-binding fragment are reclaimed and purified to outer include.

In one embodiment, the present invention additionally provides a kind of above-mentioned antibody or its antigen binding fragment being used in medicine Section.In one embodiment, purposes is treatment inflammatory disease, autoimmune disease, respiratory disease, dysbolism or cancer Disease.In one embodiment, purposes (Crohn's disease (Crohn's disease), is burst for treatment psoriasis, inflammatory bowel disease Ulcer colitis), psoriatic arthritis, multiple sclerosis, rheumatoid arthritis or ankylosing spondylitis.In an embodiment party In case, purposes is treatment psoriasis.In one embodiment, purposes is treatment inflammatory bowel disease.

In one embodiment, the present invention additionally provides one kind to include above-mentioned antibody molecule or antigen-binding fragment and medicine The pharmaceutical composition of acceptable supporting agent on.

In one embodiment, the present invention additionally provides one kind to treat inflammatory disease, autoimmune disease, respiratory system The method of disease, dysbolism or cancer, it includes giving the above-mentioned anti-IL- of effective dose to individual in need, such as patient 23p19 antibody or its antigen-binding fragment or the pharmaceutical composition comprising the antibody or its antigen-binding fragment.In an implementation In scheme, antibody or antigen-binding fragment are given by parenteral administration approach or through intravenously or subcutaneously giving.In a reality Apply in scheme, antibody or antigen-binding fragment are through subcutaneous administration.In one embodiment, the disease is psoriasis, inflammatory Enteropathy (Crohn's disease, ulcerative colitis), psoriatic arthritis, multiple sclerosis, rheumatoid arthritis or tatanic Spondylitis.In one embodiment, the disease is psoriasis.In one embodiment, the disease is inflammatory bowel disease.

In one embodiment, the present invention additionally provides the IL-23 on a kind of suppression IL-23 and mammalian cell by The method that body combines, it includes giving above-mentioned antibody molecule or antigen-binding fragment to the cell, thus suppressed by DCRS5 The signal transduction of mediation.

In one embodiment, the individual method with IL-23 associated conditions is treated the present invention additionally provides a kind of, It includes giving above-mentioned antibody or antigen-binding fragment to the individual, and the antibody or antigen-binding fragment are combined with mankind IL-23.

In one embodiment, detect and/or quantitative determine the IL-23 in biological sample the present invention additionally provides a kind of The method of content, its by the sample is contacted with above-mentioned antibody or antigen-binding fragment and detect the antibody or its fragment with IL-23p19 with reference to and reach.This information can be used for diagnosis IL-23 associated conditions.It thus provides for diagnosing IL-23 phases Related disorders determine that the whether increased method of risk of IL-23 associated conditions occurs in individual, and wherein this method includes making from individual The biological sample of body contacts with above-mentioned antibody or antigen-binding fragment, and detects the antibody or antigen-binding fragment and IL-23p19 Combination to determine IL-23 expression or concentration.

In one embodiment, combined the present invention additionally provides a kind of suppression IL-23 with the DCRS5 on cell Method, it includes giving above-mentioned antibody or antigen-binding fragment to the cell or cellular environment, thus suppresses to be situated between by DCRS5 The signal transduction led.

Brief description

Fig. 1：The comparison of mouse variable region and humanization variable region.Fig. 1 a：Vk areas engineered anti-IL-23 p 19 6B8. Fig. 1 b：VH areas engineered anti-IL-23 p 19 6B8.

Amino acid is numbered according to standard K abat numberings flow.Normal font=the mankind；Italic/underline font= Muroid；Shade font=synthesis；Runic/italic/underlines=CDR.

Fig. 2：The competitive binding assay that mankind IL-23 is combined with IL-23R/Fc.

Detailed description of the invention

IL-23 p19 subunits (being also known as " IL-23p19 " and " p19 subunits " herein) have 189 to be a kind of The polypeptide of individual amino acid, it contains targeting sequencing (Oppmann et al., the Immunity 13 with 21 amino acid:715 (2000), SEQ ID NO:181).The bioactivity of the molecule is only when it arranges in pairs or groups to form IL-23 with IL-12p40 subunits Just it is detected.IL-23 is mainly expressed by dendritic cell activated (DC) and phagocyte.It was found that IL-23 acceptor is by IL-12 The subunits of IL-12R β 1 of acceptor and referred to as IL-23R unique subunit collocation form (Parham et al., J.Immunol.168:5699(2002)).Detect that this receptor is mainly expressed on memory T cell and NK cells.Therefore, this is thin Intracellular cytokine:The expression of acceptor pair seems to be confined to certain immune cells colony.Although it is initially considered that IL-12 and IL-23 will be shared Many functions, but data show it is really not so.Although IL-12 has main function in the generation of Th1 cells, IL- is found 23 be critically related in assert recently be referred to as Th17 Th cell subsets generation and maintenance in (Kikly et al., Curr.Opin.Immunol.18:670(2006)；Kastelein et al., Ann.Rev.Immunol.25:221(2007)).This A little cells produce IL-17A, IL-17F, IL-22 and other pro-inflammatory cytokines, such as IL-6 and TNF-α.As described below, close Show it as the driving force in chronic inflammation and LADA in the Research of Animal Model for Study of the effect of these Th17 cells Importance.

The present invention provides the p19 subunits with IL-23, the antibody that particularly mankind IL-23p19 is combined.The present invention also relates to And the humanized antibody of identification IL-23 p19 subunits.In a particular embodiment, the sequence of these humanized antibodies base Identified in the sequence of some leading mouse antibodies.

The leading mouse antibodies of the present invention are produced by mouse fusion knurl.Immunity inoculation is carried out to mouse using different technologies. For example, have to mankind IL-23p19 albumen or its fragment specific antibody can be directed to such as separation IL-23p19 albumen, A part (including synthetic peptide) for the IL-23 albumen of separation, the hybridization IL-23 albumen of separation and/or any above protein Immunogenic antigens and produce.For example, use the hybridization comprising hyperkine p40 subunits and mankind's IL-23p19 subunits IL-23 protein carries out immunity inoculation to mouse.It any suitable technology as known in the art can be used to prepare immunogenicity to resist Former and generation monoclonal antibody.

Mankind IL-23 high-affinity is selected it based on leading mouse antibodies.Therefore, in an aspect, The present invention provides a kind of antibody combined with high-affinity with mankind IL-23.Selected mouse antibodies are through humanization to produce people source Change antibody.The humanized antibody of the present invention is combined with high-affinity with mankind IL-23.Therefore, in another aspect, the present invention carries For a kind of humanized antibody combined with high-affinity with mankind IL-23.

Therefore, in one embodiment, the present invention provides a kind of K_DAnti-il-23 p 19 antibodies less than 40pM.Another In individual embodiment, the present invention provides a kind of K_DAnti-il-23 p 19 antibodies less than 20pM.In another embodiment, this hair It is bright that a kind of K is provided_DAnti-il-23 p 19 antibodies less than 10pM.In another embodiment, the present invention provides a kind of K_DIt is less than 1pM anti-il-23 p 19 antibodies.

In another aspect, it is not present or in the presence of 50% human serum in human serum, antibody of the invention is with height Affinity is combined with IL-23p19.

In another aspect, humanized antibody of the invention is also combined with high-affinity with stump-tailed macaque IL-23.

In another aspect, antibody of the invention is combined with IL-23, but is not combined with IL-12.In another aspect, originally The antibody of invention does not disturb the IL-12 of the closely related family member as IL-23 bioactivity.

In another aspect, antibody of the invention suppresses mouse boosting cell stimulates generation IL-17 through IL-23.

In another aspect, humanized antibody of the invention suppresses the STAT3 phosphorylations induced in DB cells by IL-23.

In another aspect, humanized antibody of the invention is combined come antagonism IL-23 by the p19 subunits with IL-23 Effect, this is acted through measured by suppression to such as IL-17 and IL-22 cell factor, and (cell factor is by IL-23 Stimulate and produce), and detected by the content reduction of these cell factors,.

In another aspect, humanized antibody of the invention has favourable pharmacokinetics (PK) overview, such as by short Illustrated in vivo half-life in tail monkey.

In another aspect, Humanized monoclonal anti-il-23 p 19 antibodies of the invention have favourable biophysics Matter, such as quality, stability or dissolubility.

In an aspect, anti-il-23 p 19 antibodies are humanized antibody.In an aspect, anti-il-23 p 19 antibodies are Monoclonal antibody.In an aspect, anti-il-23 p 19 antibodies are full length antibody.In an aspect, anti-il-23 p 19 antibodies For Humanized monoclonal antibodies, such as total length Humanized monoclonal antibodies.

The antibody or its antigen-binding fragment of the present invention identifies specific " IL-23p19 epitopes " or " IL-23p19 tables Position ".As used herein, these terms are to refer to and the molecule of anti-il-23 p 19 antibodies immune response (such as peptide) or molecule piece Section and the IL-23p19 antigenic determinants for example identified including any antibody by being combined with following light chain/sequence of heavy chain：SEQ ID NO:84/121、86/123、88/125、90/127、91/128、93/130、95/132、97/134、99/136、101/138、 103/140、105/142、107/144、109/146、111/148、113/150、115/152、117/154、119/156、160/ 166th, 160/168,158/166 or 158/168.IL-23p19 epitopes may include protein, protein fragments, peptide or its In analog.Epitope most commonly protein, short oligopeptides, oligopeptide mimics (that is, simulation IL-23p19 antigens antibody binding The organic compound of property) or its combination.Think the peptide of antibody or the minimum dimension of polypeptide epitope is about four to five amino acid. Peptide or polypeptide epitope contain for example, at least seven amino acid or for example, at least nine amino acid or e.g., from about 15 to about 20 amino Acid.Because the recognizable Antigenic Peptide or polypeptide in three-level form of antibody, the amino acid for forming epitope needs not be continuous, And in some cases may be not even on same peptide chain.Epitope can pass through various technologies determinations as known in the art, example As X-ray crystallography, hydrogen/deuterium exchange mass spectrum (HXMS), direct mutagenesis, alanine scanning mutagenesis and peptide screening method.

The vague generalization structure of antibody or immunoglobulin is well known to those skilled in the art.These molecules is normally about The miscellaneous four glycan albumen of 150,000 dalton (dalton), be made up of two identical light (L) chains and two identical heavy (H) chains and Commonly referred to as full length antibody.Each light chain is covalently attached to form heterodimer, and miscellaneous four dimeric molecule by a disulfide bond and heavy chain Covalent disulfide bonds between two identical heavy chains of heterodimer are formed.Although light chain is with heavy chain by a disulfide bond Together, but the disulfide bond number between two heavy chains changes with Immunoglobulin Isotype.Each heavy chain and light chain also have The intrachain disulfide bridges of aturegularaintervals.Each heavy chain has variable domain (V in aminoterminal_H), it is then three or four constant domain (C_H1、 C_H2、C_H3And C_H4) and between C_H1With C_H2Between hinge area.Each light chain has two domains, i.e. aminoterminal variable domain (V_L) and carboxylic Cardinal extremity constant domain (C_L)。V_LDomain and V_HDomain noncovalent associations, and C_LDomain is generally via disulfide bond and C_H1Domain is covalently attached.It is it is believed that special Determine amino acid residue formed between light chain and heavy chain variable domain interface (Chothia et al., 1985, J.Mol.Biol.186: 651-663).Variable domain is also known as variable region herein.

Some domains in variable domain are rather different between different antibodies, are also " height becomes ".These high variable domains contain Directly participate in the residue in combination and specificity of each specific antibodies to its specific antigen determinant.Light chain and heavy chain variable domain two The denatured of person is concentrated in three sections of referred to as complementary determining region (CDR) or hypervariable loop (HVL).CDR passes through Kabat etc. People, 1991, Sequences of Proteins of Immunological Interest, the 5th edition, Public Health Sequence in Service, National Institutes of Health, Bethesda, Md. compares determination, and HVL is (at this It is also known as CDR in text) structure determined according to the three-dimensional structure of variable domain, such as by Chothia and Lesk, 1987, J.Mol.Biol.196:Described in 901-917.This two methods can cause the identification to CDR to be slightly different.As by Kabat institute really Fixed, CDR-L1 is located at the about residue 24-34 in light-chain variable domain, and CDR-L2 is located at about at residue 50-56, and CDR-L3 is located at About at residue 89-97；CDR-H1 is located at the about residue 31-35 in heavy chain variable domain, and CDR-H2 is located at about at residue 50-65, And CDR-H3 is located at about at residue 95-102.Covering specific CDR exact residue numbers will become depending on CDR sequence and size Change.Those skilled in the art can be in view of the variable region amino acid sequence of antibody routinely determines which residue forms specific CDR.Cause This, CDR1, CDR2, CDR3 of heavy chain and light chain define unique functional properties specific to given antibody.

Three CDR in each heavy chain and light chain to be spaced by skeleton area (FR), skeleton area contain changeability generally compared with Low sequence.From the amino terminal of heavy chain and light-chain variable domain to carboxyl terminal, FR and CDR are arranged in the following sequence：FR1、 CDR1, FR2, CDR2, FR3, CDR3 and FR4.FR larger beta sheet configuration make the CDR in each chain be closely adjacent to each other and with it is another CDR in chain close to.Gained conformation contributes to antigen binding site (referring to Kabat et al., 1991, NIH Publ. 91- No. 3242, I volume, the 647-669 pages), but and not all CDR residues all must directly participate in antigen binding.

FR residues and Ig constant domains do not participate in antigen binding directly, but contribute to antigen binding and/or mediate antibody to imitate Answer subfunction.It is believed that some FR residues to antigen binding at least in a manner of three kinds to produce active effects：It is directly non-with epitope Covalent bond, interacted with one or more CDR residues, and influence the interface between heavy chain and light chain.Constant domain is not direct Antigen binding is participated in, but mediates various Ig effector functions, such as mediate antibody participation antibody-dependent cytotoxicity (ADCC), Complement-dependent cytotoxicity (CDC) and antibody dependent cellular phagocytosis (ADCP).

Can the amino acid sequence based on constant domain by the light chain of vertebrate immune globulin distribute to two it is dramatically different Species (κ and λ) in one kind.By comparing, the heavy chain of mammalian immunoglobulin is divided into according to the sequence of constant domain Five primary categories below：IgA, IgD, IgE, IgG and IgM.IgG and IgA are further divided into subclass (isotype), such as IgG₁、IgG₂、IgG₃、IgG₄、IgA₁And IgA₂.Corresponding to different immune globulin classes heavy-chain constant domains be referred to as α, δ, ε, γ and μ.The subunit structure and 3-d modelling of each native immunoglobulin species are known from.

Term " antibody ", " anti-il-23 p 19 antibodies ", " humanization anti-il-23 p 19 antibodies ", " humanization anti-IL-23 p 19 Epitope antibodies " and " variant humanization anti-IL-23 p 19 epitope antibodies " specifically cover monoclonal antibody, and (including total length monoclonal resists Body), polyclonal antibody, multi-specificity antibody (for example, bispecific antibody) and expression it is expected biological activity (such as IL- 23P19 combinations) antibody fragment (such as variable domain and other parts of antibody).

Term " monoclonal antibody " (mAb) refers to high degree of specificity, i.e., for single antigenic determinat (i.e. " table Position ") there is high degree of specificity.Therefore, qualifier " monoclonal " indicator is to the antibody of same epitope, and should not be construed as needing The antibody is produced by any ad hoc approach.It should be understood that monoclonal antibody can pass through any technology known in the art or side It is prepared by method；Including (such as) hybridoma method (Kohler et al., 1975, Nature 256:495) it is, or known in the art Recombinant DNA method (for example, with reference to U.S. Patent No. 4,816,567), or pass through documents below using phage antibody library Described in technology separation in the method for monoclonal antibody caused by recombination form：Clackson et al., 1991, Nature352： 624-628；And Marks et al., 1991, J.Mol.Biol.222：581-597.

Term " monomer " refers to the homogeneous form of antibody.For example, for full length antibody, monomer refers to two identical heavy The monomeric igg of chain and two identical light chains.

Chimeric antibody is by heavy chain of antibody and light chain from a species (for example, non-human mammal, such as mouse) The heavy chain and constant region of light chain of variable region and another species (for example, people) antibody form, and can obtain in the following manner：Even Fetch the DNA sequence dna from the encoding antibody variable of the first species (for example, mouse) and the volume from the second species (such as people) The DNA sequence dna of code antibody constant region, and host is converted with containing the expression vector through catenation sequence, so that it can be produced Chimeric antibody.Or in chimeric antibody, one or more regions or domain in heavy chain and/or light chain also can be with being immunized from another Corresponding sequence in the monoclonal antibody of globulin classes or isotype is identical, homologous or for its variant or from consensus sequence Or Germline sequences.Chimeric antibody may include the fragment of the antibody, and condition is the expectation that antibody fragment gives expression to its parental generation antibody Biological activity, such as combined with same epitope (for example, with reference to U.S. Patent No. 4,816,567；And Morrison et al., 1984, Proc.Natl.Acad.Sci.USA 81：6851-6855).

Term " antibody fragment ", " anti-il-23 p 19 antibodies fragment ", " anti-IL-23 p 19 epitope antibodies fragment ", " humanization Anti-il-23 p 19 antibodies fragment ", " humanization anti-IL-23 p 19 epitope antibodies fragment ", " variant humanization anti-IL-23 p 19 epitope Antibody fragment " refers to a part for total length anti-il-23 P19 antibody, wherein retain variable region or Functional Capability, such as specificity IL-23P19 epitopes combine.The example of antibody fragment includes but is not limited to Fab, Fab', F (ab')₂, Fd, Fv, scFv and ScFv-Fc fragments, bivalent antibody (diabody), linear antibodies, single-chain antibody, miniantibody (minibody), from antibody fragment shape Into bivalent antibody and from antibody fragment formed multi-specificity antibody.

Available antibodies fragment can be generated with the ferment treatment full length antibody such as papain or pepsin.Use wood Melon protease digestion is referred to as the antigen binding antibody fragment of " Fab " fragment (each with single antigen to produce two identicals Binding site) and residual " Fc " fragment.Fab fragments also contain light-chain constant domains and heavy chain C_H1Domain.Pepsin produces F (ab')₂Fragment, it has two antigen binding sites and remains to crosslinking antigen.

The difference of Fab' fragments and Fab fragments is in C_H1Additional residue, including one or more be present in the C-terminal in domain Cysteine from antibody hinge region.F(ab')₂Antibody fragment is to be connected by the cysteine residues in hinge area Fab' fragments pair.Also other chemical couplings of known antibodies fragment.

" Fv " fragment contains intact antigen identification and binding site, and the site is by a heavy chain variable domain and a light chain Variable domain is in the dimer composition being closely not covalently linked.In this configuration, three CDR interactions of each variable domain, from And in the V_H-V_LAntigen binding site is defined on the surface of dimer.Six CDR assign the antibody antigen and combined specifically jointly Property.

" scFv " or " scFv " antibody fragment is to include antibody V_HAnd V_LThe scFv variant in domain, wherein the domain is present In Single polypeptide chain.ScFv can identify and combine antigen.ScFv polypeptides also can optionally contain and be located at V_HDomain and V_LBetween domain Peptide linker, with promote scFv formed for antigen binding expectation three-dimensional structure (for example, with reference to Pluckthun, 1994, In The Pharmacology of monoclonal Antibodies, volume 113, Rosenburg and Moore are edited, Springer-Verlag, New York, the 269-315 pages).

" bivalent antibody " refers to the small antibody fragment with two antigen binding sites, and the fragment is included in same polypeptide Heavy chain variable domain (the V connected in chain_H) and light-chain variable domain (V_L)(V_H-V_LOr V_L-V_H).Bivalent antibody is described more fully in for example Holliger et al., (1993) Proc.Natl.Acad.Sci.USA 90:In 6444-6448.

Other known antibody fragment includes those Fd sections (V for including a pair of series_H-C_H1-V_H-C_H1) to be formed a pair The antibody fragment of antigen binding domain." linear antibodies " can be bispecific or monospecific, such as (e.g.) Zapata et al., 1995, Protein Eng.8 (10):Described in 1057-1062.

" humanized antibody " or " humanized antibody fragment " is certain types of chimeric antibody, and it includes immunoglobulin ammonia Base sequence variants or its fragment, it can combine predetermined antigens, and it substantially has human immunoglobulin(HIg) comprising one or more The FR of amino acid sequence and one or more substantially CDR with non-human immunoglobulin amino acid sequence.This non-human amino acid Sequence commonly referred to as " inputs " sequence, is normally taken from " inputting " antibody domain, especially variable domain.Generally, humanized antibody is at least CDR or HVL including non-human antibody, insert between people's heavy chain or the FR of light-chain variable domain.The present invention illustrates that specific humanization resists IL-23P19 antibody, it contains is derived from muroid shown in the table 3 and 4 between insertion human germ line sequences' heavy chain and the FR of light-chain variable domain The CDR or humanization CDR of monoclonal antibody.It should be understood that some muroid FR residues may be important to the function of humanized antibody, and Therefore some human germ line sequences' heavy chains and light-chain variable domain residue will be modified, with identical with those in corresponding muroid sequence.

In another aspect, it is variable to basically comprise all at least one (usual two) for humanization anti-il-23 P19 antibody Domain (such as being contained in person in such as Fab, Fab', F (ab') 2, Fabc and Fv fragments), wherein all or substantially all CDR are corresponding In the CDR of non-human immunoglobulin, and all CDR are particularly herein by the muroid sequence that is described in detail in this paper table 1 below to 4 Or humanized sequence, and all or substantially all FR are the FR of human immunoglobulin(HIg) consensus sequence or Germline sequences.Another In aspect, humanization anti-il-23 P19 antibody also includes at least one of immunoglobulin (being usually human immunoglobulin(HIg)) Fc areas Point.Generally, antibody contains light chain and at least both heavy chain variable domains.If suitable, antibody also may include heavy chain C_H1, hinge, C_H2、C_H3And/or C_H4One or more of area.

Humanization anti-il-23 P19 antibody may be selected from any kind immunoglobulin (including IgM, IgG, IgD, IgA and ) and any isotype (including IgG IgE₁、IgG₂、IgG₃、IgG₄、IgA₁And IgA₂).For example, constant domain can be complement fixation Constant domain, wherein it is expected humanized antibody expression cell toxic activity, and isotype is usually IgG₁.If the cell is not expected Toxic activity, then constant domain can be another isotype, such as IgG₂.Alternative humanization anti-il-23 P19 antibody can be included and come from The sequence of more than one immune globulin classes or isotype, and those skilled in the art can skillfully select particular constant domain next excellent Change desired effector function.In a particular embodiment, antibody provided by the present invention is IgG1 antibody, and more specifically To reject the IgG1 antibody of effector function.

The FR and CDR or HVL of humanization anti-il-23 P19 antibody need not be accurately corresponding with parental sequences.For example, it can lead to Cross substitution, insertion or lack change (for example, mutagenic treatment) input CDR or HVL or shared FR or germline FR sequences in one Or multiple residues, so that gained amino acid residue is no longer identical with the Original Residue of correspondence position in any parental sequences, But antibody still retains the function of being combined with IL-23P19.The change generally not can extensively change and can be conservative change.Generally, Those residues that at least 75% humanized antibody residue can share FR or germline FR with parental generation and input in CDR sequence are corresponding, more Generally have at least 90% and most commonly have more than 95% or the residue correspondence more than 98% or more than 99%.

Influence the interface (" V between heavy chain and light chain variable district_L-V_HInterface ") immunoglobulin residues be those influence The residue that two chains are approached or oriented relative to each other.Some residues that may participate in interchain interaction include V_LResidue 34, 36th, 38,44,46,87,89,91,96 and 98 and V_HResidue 35,37,39,45,47,91,93,95,100 and 103 (uses Kabat et al., Sequences of Proteins of Immunological Interest (National Institutes Of Health, Bethesda, Md., 1987) described in numbering system).U.S. Patent No. 6,407,213 is also discussed, example Such as V_LResidue 43 and 85 and V_HThe grade residue of residue 43 and 60 may also participate in this interaction.Although illustrate institute only for human IgG Residue is stated, but it is also applied to other species.Selection reasonably expects that the important antibody residue for participating in interchain interaction is used for Substitution in consensus sequence.

Term " consensus sequence " and " shared antibody " refer in all of any particular types, isotype or subunit structure Each position in immunoglobulin (such as human immunoglobulin(HIg) variable domain) includes the amino of the amino acid residue most often occurred Acid sequence.Consensus sequence can be based on particular species or multiple species immunoglobulin." shared " sequence, structure or antibody can be managed Solve to cover the shared human sequence described in some embodiments, and refer in any particular types, isotype or subunit knot Each position in all human immunoglobulin(HIg)s of structure includes the amino acid sequence of the amino acid residue most often occurred.Therefore, altogether There is the amino acid sequence contained by sequence that there is the amino acid being present in one or more known immunoglobulins in each position, But it and may inaccurately replicate the whole amino acid sequence of any single immunoglobulin.Variable region consensus sequence is not appointed certainly What naturally-produced antibody or immunoglobulin and its variant can obtain (Kabat et al., 1991, Sequences of Proteins of Immunological Interest, the 5th edition, Public Health Service, National Institutes of Health, Bethesda, Md.).Heavy chain and the FR of light chain consensus sequence and its variant resist for humanization The preparation of IL-23P19 antibody provides available sequences.For example, with reference to U.S. Patent No. 6,037, No. 454 and the 6th, 054, No. 297.

Human germ line sequences are naturally occurring in ethnic group.The combination of the germ line genes produces antibody diversity.Antibody is light The germline antibody sequence of chain comes from conservative people's germline κ or λ v- genes and j- genes.Similarly, sequence of heavy chain comes from germline v-, d- And j- genes (LeFranc, M-P and LeFranc, G, " The Immunoglobulin Facts Book " Academic Press, 2001).

As used herein, " variant ", " anti-IL-23 p 19 variant ", " humanization anti-IL-23 p 19 variant " or " variant people source Change anti-IL-23 p 19 " each refer at least have the light chain variable murine CDR sequence from any sequence as shown in table 1, Or at least there is the humanization anti-IL-23 p 19 of the heavy chain murine CDR sequence for coming from murine monoclonal antibody as shown in table 2 Antibody.Variant is included in the variant in one or two light chains or heavy chain variable domain with the change of one or more amino acid, and it is limited Condition be amino acid change will not substantial damage antibody and IL-23p19 combination.Exemplary humanization caused by herein resists Body includes referred to as antibody A, antibody B, antibody C and antibody D antibody, and the various light chains and heavy chain of the antibody are showed in respectively SEQ ID No:174 and 180 and SEQ ID No:In 176 and 178.

" separation " antibody is antibody that is identified and separating and/or reclaim from its natural surroundings component.Antibody Groups of contaminants in natural surroundings is divided into the material of those diagnostic or therapeutic applications that may interfere with antibody, and can be enzyme, swash Element or other Proteinaceous or non-protein solutes.In an aspect, can be by antibody purification to separating degree is extremely in terms of antibody weight It is more than 95% less.

Because at least one of antibody natural surroundings component is not in, therefore the antibody separated includes producing its restructuring Intracellular antibody iM situ.However, the antibody of separation can generally be prepared by least one purification step, wherein removing weight Group cellular material.

Term " antibody performance " refers to contribute to the factor of antibody identification antigen or antibody in vivo effect.Antibody amino groups The change of acid sequence can influence the antibody characteristic such as folding, and can influence for example following physical factor：Antibody and antigen knot Initial rate (the k of conjunction_a), the dissociation constant (k of antibody and antigen_d), antibody is to the affinity costant (Kd), antibody conformation, egg of antigen White stability and antibody half life.

Terms used herein " Epitope tag " refers to that anti-il-23 P19 antibody merges with " epitope tag "." epitope tag " To provide the polypeptide of epitope for antibody generation with enough amino acid, and it is designed such that it does not disturb humanization to resist The expectation activity of IL-23P19 antibody.Generally there is epitope tag fully uniqueness to be resisted with to be directed to caused by the epitope tag Body substantially will not be with other epitope cross reactions.Suitable tag polypeptide typically contain at least six amino acid residue and generally Contain about 8 to 50 amino acid residues or about 9 to 30 residues.The example of epitope tag and the antibody of combination epitope includes influenza HA tag polypeptides and its antibody 12CA5 (Field et al., 1988Mol.Cell.Biol.8：2159-2165)；C-myc labels and Its 8F9,3C7,6E10, G4, B7 and 9E10 antibody (Evan et al., 1985, Mol.Cell.Biol.5 (12):3610-3616)； And herpes simplex virus glycoprotein D (gD) labels and its antibody (Paborsky et al., 1990, Protein Engineering 3 (6)：547-553).In certain embodiments, epitope tag is " salvage receptor binding epitope ".Terms used herein " is remedied Receptor binding domain (salvage receptor binding epitope) " refers to IgG molecules (such as IgG₁、IgG₂、IgG₃ Or IgG₄) epitope in Fc areas, it is responsible for the in vivo serum half-life for extending IgG molecules.

In some embodiments, antibody of the invention can be conjugated with cytotoxic agent.Cytotoxic agent is suppression or resistance Only cell function and/or cause any material of cytoclasis.The term is intended to include radio isotope (such as I¹³¹、I¹²⁵、 Y⁹⁰And Re¹⁸⁶), chemotherapeutic agent and toxin (such as bacterium, fungi, plant or animal sources enzyme activity toxin) and its fragment.Institute Stating cytotoxic agent can be coupled by standard operation and the humanized antibody of the present invention, and its can be used for (such as) treatment be applicable it is anti- The patient of autogenic therapy.

" chemotherapeutic agent " is the chemical compound available for treating cancer.Chemotherapeutic agent has a variety of examples can be with Therapeutic antibodies of the present invention are conjugated.The example of the chemotherapeutic agent includes alkylating agent, for example, thio-tepa (thiotepa) and Endoxan；Alkyl sulfonate esters, such as busulfan (busulfan), Improsulfan (improsulfan) and piposulfan (piposulfan)；Aziridine, for example, benzo DOPA (benzodopa), carboquone (carboquone), meturedopa and uredopa；Ethylenimine and methyl melamine, including hexamethyl melamine (altretamine), triethylene melamine, triethylene Phosphamide, triethylenethiophosphoramide and trihydroxy methyl melamine；Annona lactone (acetogenin) (in particular bullatacin (bullatacin) and bullatacinone (bullatacinone))；Camptothecine (camptothecin) (including synthetic analogues Hycamtin (topotecan))；Bryostatin (bryostatin)；callystatin；CC-1065 (including it synthesizes class Like thing Adozelesin (adozelesin), Carzelesin (carzelesin) and Bizelesin (bizelesin))； Cryptophycin (in particular cryptophycin 1 and cryptophycin 8)；Dolastatin (dolastatin)； Auristatin (including analog monomethyl-auristatin E and monomethyl-auristatin F)；More card meter Xing (duocarmycin) (including synthetic analogues KW-2189 and CBI-TMI)；Ai Liusu (eleutherobin)； pancratistatin；sarcodictyin；spongistatin；Nitrogen mustards, such as Chlorambucil (chlorambucil), It is Chlornaphazine (chlornaphazine), chlorine phosphamide (chlorophosphamide), Estramustine (estramustine), different Phosphamide (ifosfamide), chlormethine (mechlorethamine), mustron (mechlorethamine Oxide hydrochloride), melphalan (melphalan), novoembichin (novembichin), phenesterin (phenesterine), prednimustine (prednimustine), Trofosfamide (trofosfamide) and uracil mustard (uracil mustard)；Nitro ureas, such as BCNU (carmustine), chlorozotocin (chlorozotocin), good fortune Mo Siting (fotemustine), lomustine (lomustine), Buddhist nun not STING (nimustine) and Ranimustine (ranimnustine)；Antibiotic, such as enediyne (enediyne) antibiotic (such as calicheamycin (calicheamicin), Especially calicheamycin γ 1I and calicheamycinSee, for example, Angew Chem.Intl.Ed.Engl. (1994) 33:183- 186)；Dynemicin, including dynemicin A；Diphosphonate, such as clodronate (clodronate)；Ai Sipeila mycins (esperamicin)；Neocarzinostatin chromophore (neocarzinostatin chromophore) and related chromoprotein alkene two Alkynes antibiotic chromophore (related chromoprotein enediyne antibiotic chromophore), Aclacinomysin classes, D actinomycin D (actinomycin), authramycin, azaserine (azaserine), it is rich come Mycin (bleomycin), act-C (cactinomycin), carabicin, carminomycin (carminomycin), thermophilic cancer Mycin (carzinophilin), chromomycin (chromomycin), actinomycin D (dactinomycin), daunorubicin (daunorubicin), Detorubicin (detorubicin), 6- diazo -5- oxn-l-norieucins (6-diazo-5- Oxo-L-norleucine), Doxorubicin (Adriamycin^TM) (including morpholino-Doxorubicin (morpholino- Doxorubicin), Cyanomorpholino-Doxorubicin (cyanomorpholino-doxorubicin), 2- pyrrolins subbase-more It is soft than star (2-pyrrolino-doxorubicin), deoxidation Doxorubicin (deoxydoxorubicin)), epirubicin (epirubicin), esorubicin (esorubicin), idarubicin (idarubicin), marcellomycin (marcellomycin), mitomycin (mitomycin) class such as mitomycin C (mitomycin C), Mycophenolic Acid (mycophenolic acid), nogalamycin (nogalamycin), olivomycin (olivomycin), Peplomycin (peplomycin), porfiromycin (porfiromycin), Puromycin (puromycin), triferricdoxorubicin (quelamycin), rodorubicin (rodorubicin), broneomycin (streptonigrin), streptozotocin (streptozocin), tubercidin (tubercidin), ubenimex (ubenimex), neoearcinostain And zorubicin (zorubicin) (zinostatin)；Antimetabolite, such as methopterin (methotrexate) and 5- fluorine urine Pyrimidine (5-fluorouracil) (5-FU)；Folacin, such as denopterin (denopterin), methopterin, sieve of talking endlessly Purine (pteropterin) and Trimetrexate (trimetrexate)；Purine analogue, such as fludarabine (fludarabine), 6-MP (6-mercaptopurine), thiapurine (thiamiprine) and thioguanine (thioguanine)；Pyrimidine Analog, such as ancitabine (ancitabine), azacitidine (azacitidine), 6- aza uridines (6- Azauridine), Carmofur (carmofur), cytarabine (cytarabine), di-deoxyuridine (dideoxyuridine), Doxifluridine (doxifluridine), Yi Nuota shores (enocitabine) and floxuridine (floxuridine)；Androgen, Such as Calusterone (calusterone), dromostanolone propionate (dromostanolone propionate), epithioandrostanol (epitiostanol), Mepitiostane (mepitiostane) and Testolactone (testolactone)；Anti-adrenergic (anti- ), such as aminoglutethimide (aminoglutethimide), mitotane (mitotane) and Trilostane adrenal (trilostane)；Folic acid supplement, such as folinic acid (frolinic acid)；Aceglatone (aceglatone)；Aldehyde phosphorus Acid amides glucosides (aldophosphamide glycoside)；Amino-laevulic acid (aminolevulinic acid)；Grace urine is phonetic Pyridine (eniluracil)；Amsacrine (amsacrine)；bestrabucil；Bisantrene (bisantrene)；She kills up to song (edatraxate)；Defosfamide (defofamine)；Demecolcine (demecolcine)；Diaziquone (diaziquone)； elfornithine；Elliptinium Acetate (elliptinium acetate)；Epothilones (epothilone)；Ethoglucid (etoglucid)；Gallium nitrate (gallium nitrate)；Hydroxycarbamide (hydroxyurea)；Lentinan (lentinan)；Chlorine Ni Daming (lonidainine)；Maytansinol (maytansinoid) class, such as maytansine (maytansine) and ansamitocin (ansamitocin)；Mitoguazone (mitoguazone)；Mitoxantrone (mitoxantrone)；mopidanmol；Rhizobium Agent (nitraerine)；Pentostatin (pentostatin)；Phenamet (phenamet)；THP (pirarubicin)；Losoxantrone (losoxantrone)；Podophyllic acid (podophyllinic acid)；2- ethyl hydrazines (2- ethylhydrazide)；Procarbazine (procarbazine)；Razoxane (razoxane)；Rhizomycin (rhizoxin)；Sizofiran (sizofiran)；Spirogermanium (spirogermanium)；Tenuazonic acid (tenuazonic acid)；Triethyleneiminobenzoquinone (triaziquone)；2,2 ', 2 "-trichlorotriethylamine (2,2 ', 2 "- trichlorotriethylamine)；Trichothecene (trichothecene) (especially T-2 toxin, verracurin A, Roridine A (roridin A) and anguidine)；Urethane (urethan)；Eldisine (vindesine)；Dacarbazine (dacarbazine)；Mannomustine (mannomustine)；Dibromannitol (mitobronitol)；Mitolactol (mitolactol)；Pipobroman (pipobroman)；gacytosine；Cytarabine (arabinoside) (" Ara-C ")； Endoxan；Phosphinothioylidynetrisaziridine；Taxane (taxoid), such as taxol (paclitaxel)Bristol-Myers Squibb Oncology, Princeton, N.J.) and Docetaxel (doxetaxel)Rhone- Poulenc Rorer, Antony, France)；Chlorambucil；Gemcitabine (gemcitabine) (Gemzar^TM)；6- sulphur birds Purine；Purinethol；Methopterin；Platinum analogs, such as cis-platinum (cisplatin) and carboplatin (carboplatin)；Vincaleukoblastinum (vinblastine)；Platinum；Etoposide (etoposide) (VP-16)；Ifosfamide；Mitoxantrone；Vincristine (vincristine)；Vinorelbine (vinorelbine) (Navelbine^TM)；Novantrone (novantrone)；Teniposide (teniposide)；Edatrexate (edatrexate)；Daunorubicin (daunomycin)；Aminopterin (aminopterin)；It is uncommon Luo Da (xeloda)；Ibandronate (ibandronate)；CPT-11；Topoisomerase enzyme inhibitor RFS 2000；Difluoromethyl Ornithine (DMFO)；Retinoid, such as retinoic acid；Capecitabine (capecitabine)；And any one in said medicine Pharmaceutically acceptable salt, acid or derivative.Also include for the anti-of the effect of regulation or inhibitory hormone to tumour in this definition Hormone preparation, such as antiestrogenic and SERM (SERM), including (such as) TAM (tamoxifen) (including Nolvadex^TM), Raloxifene (raloxifene), Droloxifene (droloxifene), 4- hydroxyls TAM, Trioxifene (trioxifene), keoxifene, LY117018, Onapristone (onapristone) and support are auspicious Meter Fen (toremifene) (Fareston^TM)；Suppress the aromatase inhibitor of aromatase enzyme, it adjusts the production of estrogen in adrenal gland It is raw, such as 4 (5)-imidazoles, amine Rumi are special, megestrol acetate (megestrol acetate) (Megace^TM), Exemestane (exemestane), formestane (formestane), Fadrozole (fadrozole), Vorozole (vorozole) (Rivisor^TM)、 Letrozole (letrozole) (Femara^TM) and Anastrozole (anastrozole) (Arimidex^TM)；And antiandrogen, such as Flutamide (flutamide), Nilutamide (nilutamide), Bicalutamide (bicalutamide), Leuprorelin And Goserelin (goserelin) (leuprolide)；And the pharmaceutically acceptable salt of any one in said medicine, acid or spread out Biology.Any one or more in the medicine can be conjugated with the humanized antibody of the present invention can be used for treating various diseases to provide The medicine of disease.

Also antibody can be made to be conjugated with prodrug." prodrug " is the precursor or derivative form of pharmaceutically active substance, itself and parent Medicine is compared smaller to the cytotoxicity of tumour cell, and enzymatic activation or can be converted into the stronger form of activity.For example, with reference to Wilman, 1986, " Prodrugs in Cancer Chemotherapy ", Biochemical Society Transactions, 14, the 375-382 pages, 615th Meeting Belfast；And Stella et al., 1985, “Prodrugs：A Chemical Approach to Targeted Drug Delivery ", Directed Drug Delivery, Borchardt et al. (editor), the 247-267 pages, Humana Press.Available prodrug includes (but unlimited In) prodrug containing phosphate radical, the prodrug containing D2EHDTPA root, the prodrug containing sulfate radical, the prodrug containing peptide, through D- ammonia Base acid modification prodrug, glycosylated prodrugs, the prodrug containing beta-lactam, containing the phenoxy-acetamide being optionally substituted before Medicine and prodrug, 5-flurocytosine prodrug and other 5-FUD prodrugs containing the phenyl-acetamides being optionally substituted, it can turn Chemical conversion has higher active no cytotoxicity medicine.Can derive the cytotoxic drug for prodrug forms example include (but It is not limited to) those chemotherapeutic agents described above.

For diagnostic and therapeutic monitoring purpose, can also make the antibody of the present invention and mark (individually mark or Mark and other the second medicine (prodrug, chemotherapeutic agent etc.)) it is conjugated.Mark is different from other second medicines, and it refers to Detectable compound or composition medicine, and it can directly or indirectly be conjugated with the humanized antibody of the present invention.Mark is in itself It can detect (for example, labelled with radioisotope or fluorescence labeling), or in the case of enzyme mark, mark can be catalyzed substrate Detectable chemical change occurs for compound or composition.Labeled humanization anti-il-23 P19 antibody can be prepared and answered for various In, including in vitro and in vivo diagnostics.

The antibody of the present invention can be allocated as the part of Liposomal formulation to realize its vivo Delivery." liposome " is The vesicles being made up of various types of lipids, phosphatide and/or surfactant.Liposome can be used for mammal deliveringization Compound or composite, such as humanization anti-il-23 P19 antibody disclosed herein, the antibody are optionally lived with one or more medicines Property medicine and/or mark coupling or combination.The component of liposome is generally arranged with double-decker, this lipid arrangement with biomembrane It is similar.

The some aspects of the present invention are related to the nucleic acid of separation, and it encodes one or more domains of the humanized antibody of the present invention." point From " nucleic acid molecules are the nucleic acid molecules identifying and separate from least one pollutant nucleic acid molecules, it is in the day of antibody nucleic acids Generally it is combined in right source with the pollutant nucleic acid molecules.The nucleic acid molecules of separation and the nucleic acid point being present in n cell It is sub different.

In each side of the present invention, one or more domains of humanized antibody are recombinantly expressed.The recombination expression can use one or Various control sequence, i.e., the polynucleotide sequence needed for the coded sequence being operatively connected is expressed in specific host organism. Control sequence suitable for prokaryotic include (such as) promoter, operator and ribosome bind site sequence.Eucaryon control Sequence processed includes but is not limited to promoter, polyadenylation signal and enhancer.The control sequence can be used in protokaryon and Humanization anti-il-23 P19 antibody is expressed and produced in eukaryotic host cell.

When making nucleotide sequence with another nucleotide sequence there is feature to contact, the nucleotide sequence passes through " being operatively connected ". For example, if presequence or secretion targeting sequencing are expressed as participating in the preceding albumen of polypeptide secretion, the presequence or the leading sequence of secretion Arrange and be operatively connected with encoding the nucleic acid of the polypeptide；If promoter or enhancer influence the transcription of the sequence, the promoter or Enhancer is operatively connected with the sequence；Or if the positioning of ribosome bind site can promote to translate, the ribosome binding site Point is operatively connected with the coded sequence.Generally, " it is operatively connected " and means that connected DNA sequence dna is continuous sequence, and is dividing Secrete under targeting sequencing situation for continuous sequence and in reading frame.However, enhancer is optionally continuous.Can be by convenient Restriction site at engagement complete to connect.If the site is not present, synthetic oligonucleotide adapter or joint can be used.

Terms used herein " cell ", " cell line " and " cell culture " is used interchangeably, and all titles are all Including its filial generation.Therefore, " transformant " and " transformed cells " includes primary individual cells and its derivative culture without considering Transfer number.

Term " mammal " for therapeutic purposes refers to any animal for being classified as mammal, including people, domestic animal And farm-animals, and zoo animal, motion use animal or pet, such as dog, horse, cat, ox etc..Preferably, mammal For people.

" illness " used herein is that can benefit from any disease using humanization anti-il-23 P19 Antybody therapies described herein Condition.The term includes chronic and acute disease or disease, including those make mammal be susceptible to suffer from the pathological condition of the illness. The non-limiting examples or illness to be treated herein include inflammatory, angiogenic, LADA and immune conditions, breathing System condition, cancer, hematologic malignancies, benign and malignant tumour, leukaemia and lymphoid malignancy.

Term " cancer " and " carcinous " refer to or described the physiology that dysregulated cellular growth is generally characterised in that in mammal Learn the patient's condition.The example of cancer includes but is not limited to carcinoma, lymthoma, blastoma, sarcoma and leukaemia.

As used herein, term " IL-23 associated conditions " or " IL-23 relevant diseases " refer to be facilitated by IL-23 activity and The symptom of the usual unconventionality expressions of IL-23.IL-23 associated conditions include disease of immune system and illness, such as autoimmune disorder And inflammatory disorders.The patient's condition includes but is not limited to rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), sclerderm Disease, Sjogren Cotards, multiple sclerosis, psoriasis, psoriatic arthritis, inflammatory bowel disease are (for example, ulcerative colitis And Crohn's disease), lung inflammation, asthma, ITP (ITP) and ankylosing spondylitis.

Term " intravenous infusion " refer in animal or people patient vein through greater than about 15 minutes, typically between about 30 to A period of time between 90 minutes introduces medicine.

Term " intravenous push " or " intravenously propelling " refer to give medicine into the vein of animal or people, so that Body receives medicine in about 15 minutes or shorter, general 5 minutes or shorter time.

Term " subcutaneous administration " refers to the capsule under the skin of animal or people patient, preferably between skin and substrate microstructure It is interior by introducing medicine from the relatively slow continual delivery of drug reservoir.Skin is pinched to from or is pulled away from substrate microstructure upwards to be produced Raw capsule.

Term " h inf " refers to the capsule under the skin of animal or people patient, preferably between skin and substrate microstructure It is interior through a period of time (including but not limited to 30 minutes or shorter or 90 minutes or shorter) by relatively slow from drug reservoir Continual delivery introduce medicine.Optionally, infusion can be by being subcutaneously implanted the drug delivery pump (skin of implantation animal or people patient Under skin) implement, wherein the pump is through predetermined amount of time (such as 30 minutes, 90 minutes or period across whole therapeutic scheme) Deliver the medicine of scheduled volume.

Term " subcutaneous bolus injection " refers to give medicine under the skin of animal or people patient, and wherein medicine injection delivering is shorter than About 15 minutes；In another aspect, it is shorter than 5 minutes；And in another aspect, it is shorter than 60 seconds.In another aspect, skin with Intracapsular between substrate microstructure is given, and wherein the capsule can be produced by the way that skin to be pinched to from or is pulled away from substrate microstructure upwards.

Term " therapeutically effective amount " used refers to that one or more sanatory symptoms of institute can be alleviated or be improved to active medicine Amount.In another aspect, therapeutically effective amount refer to have shown can effectively (such as) slow down the target serum-concentration of progression of disease. It may depend on the patient's condition to be treated and measure effect in the usual way.

" treatment " used herein and " therapy " and similar terms are intended to include for the therapeutic and pre- of disease or illness Anti- property or inhibition measure, it produces any clinically expectation or beneficial effect, including but not limited to mitigates or alleviate disease One or more symptoms of disease or illness, make disease or illness regression, the progress that disease or illness is slowed or stopped.Thus, for example, Term treatment gives medicine before or after being included in the paresthesia epilepsy of disease or illness, thus prevents or eliminates a disease or illness One or more signs.As another example, the term gives medicine after being included in the clinical expression of disease, so as to resist the disease The symptom of disease.In addition, medicine is given after breaking-out and after having there are clinical symptoms includes " treatment " used herein or " therapy ", No matter wherein whether the treatment causes amelioration of disease, the clinical parameter for all influenceing disease or illness, such as tissue damage degree are given Or the amount or degree of transfer.As long as in addition, independent or the present composition combined with another medicine and unused people source Compared to mitigating or improving the sanatory at least one symptom of institute when changing IL-23P19 antibody compositions, whether mitigate the disease All symptoms of disease, the result should be regarded as effectively treating potential illness.

Term " package insert " used refers to the specification being typically included in treatment product commercial packing, and it is containing relevant In using the indication of the treatment product, usage, give, the information of contraindication and/or warning.

Antibody

In an aspect, it is described herein and discloses anti-il-23 antibodies, particularly humanization anti-il-23 p 19 antibodies；And Comprising one or more anti-il-23 antibodies, particularly the composition of one or more humanization anti-il-23 p 19 antibodies of the invention and Product.Also description includes the bonding agent of anti-il-23 antibodies, the particularly antigen-binding fragment of humanization anti-il-23 p 19 antibodies. Humanization anti-il-23 p 19 antibodies and bonding agent can suppress the generation of Th17 relevant cell factors, and Th17 relevant cell factors can promote Into chronic auto-immune and inflammatory disease.Therefore, humanization anti-il-23 p 19 antibodies and bonding agent can be used for treating a variety of diseases Or illness.Humanization anti-il-23 p 19 antibodies and IL-23p19 bonding agents each include specific recognition IL-23p19 epitopes extremely A few part (that is, antigen-binding fragment).

In initial token, mouse antibodies are selected based on IL-23p19 binding characteristics.

Therefore, in an aspect, antibody of the invention is to IL-23, the particularly K to mankind IL-23_DLess than 100pM. In another aspect, the K of antibody of the invention_DLess than 40pM.In another aspect, the K of antibody of the invention_DLess than 20pM. In another aspect, the K of antibody of the invention_DLess than 10pM.In another aspect, the K of monoclonal antibody of the invention_DIt is less than 1pM。

Selected mouse antibodies have following light chain variable district and weight chain variable district as shown in table 1 and table 2.

Table 1：Anti-IL-23 p 19 mouse leader thing-VK sequences

Table 2：Anti-il-23 p29 mouse leader thing-VH sequences

It is each based on framework homology, CDR structures, conservative Typical residues, conservative interface filling residue and other parameters selection Human skeleton's sequence of mouse leader thing.

The mouse light chain and heavy chain CDR of various mouse antibodies are showed in table 3 and table 4 respectively.Table 4 is also shown via people source Change method comes from mouse antibodies 6B8 three heavy chain CDR.

Table 3：CDR sequence

Table 4：Heavy CDR sequences

Above CDR listed in table 3 and table 4 determined using Chothia numbering systems (Al-Lazikani et al., (1997)JMB 273,927-948)。

The Fab preferably or on an equal basis combined is shown compared to chimeric parent Fab, is chosen for changing into IgG.6B8 is turned It is melted into IgG1KO forms.IgG1KO (rejecting effector function) has two mutation Leu234Ala and Leu235Ala in Fc areas, It reduces effector function, such as Fc γ R and complement combine.IgG forms describe in the literature (see, for example, Hezareh et al., (2001)Journal of Virology 75:12161-12168).Humanization approach is described in more detail in embodiment 1.This person source The result of change is to produce humanized antibody sequence.Come from mouse antibodies 6B8 humanization light chain variable district and weight chain variable district Representativeness numbering is provided and is showed in table 5 and table 6.Come from mouse antibodies 6B8 humanization light chain variable district and weight chain variable Comparison between area and mouse antibodies 6B8 light chain variable district and weight chain variable district is showed in Fig. 1.

The selected combination for coming from mouse antibodies 6B8 humanization light chain variable district and weight chain variable district produces antibody A, B, C And D：

Antibody A：6B8-IgG1KO-2 (weight chain variable district 6B8CVH-02 and light chain variable district 6B8CVK- with IgK-66 66)；

Antibody B：6B8-IgG1KO-5 (weight chain variable district 6B8CVH-05 and light chain variable district 6B8CVK- with IgK-66 66)；

Antibody C：6B8-IgG1KO-2 (weight chain variable district 6B8CVH-02 and light chain variable district 6B8CVK- with IgK-65 65)；

Antibody D：6B8-IgG1KO-5 (weight chain variable district 6B8CVH-05 and light chain variable district 6B8CVK- with IgK-65 65)。

Antibody A, B, C and D have the heavy chain and sequence of light chain being showed in table 7.

Table 5：Humanization 6B8-VK sequences

Table 6：Humanization 6B8-VH sequences

Table 7：Antibody A, B, C and D heavy chain and light chain DNA and amino acid sequence

In upper table 7, antibody A, B, C and D light chain variable district and weight chain variable district underline.

In an aspect, humanization anti-il-23 p 19 antibodies of the invention have following at least one property.Another In aspect, humanization anti-il-23 p 19 antibodies of the invention have following at least two or at least 3,4,5,6,7,8,9,10 or 11 Any combinations of kind property.In another aspect, humanization anti-il-23 p 19 antibodies of the invention have following all properties.

To mankind IL-23 K_D≤ 1pM (association rate is unchanged in 50% human serum)

In vitro IL-23 is blocked to be combined with mankind IL-23R/Fc

Do not combined with human IL-12

The IL-17 for suppressing to be induced by mankind IL-23 in mouse boosting cell is produced, IC₅₀≤20pM

Suppress the STAT3 phosphorylations induced in mankind's DB cells by mankind IL-23, IC₅₀≤40pM

ADCC/CDC activity without prediction

To stump-tailed macaque IL-23 K_D≤1pM

With mouse or rat IL-23 no cross reactions

Suppress to be produced (to two kinds of cells under 1mg/kg by the mankind IL-23 IL-17 induced and IL-22 in mouse ear The suppression of the factor is >=80%)

83 DEG C of stability (83 DEG C of melting temperature, as determined by Differential Scanning Calorimetry determination method)

Solubility >=100mg/ml (is such as measured by ultraviolet spectroscopy and monitored by turbidity)

Subcutaneous administration 1.0mg/kg is shown in three stump-tailed macaques, and >=10nM exposure lasts about 28 days, biological utilisation Degree is about 70%.

Herein, the ADCC/DC activity of no prediction refers to the humanization anti-il-23 p 19 antibodies of the present invention to Fc acceptors Affinity reduce and therefore prediction without ADCC/CDC activity.

In an aspect, humanization anti-il-23 p 19 antibodies of the invention have following at least one property.Another In aspect, humanization anti-il-23 p 19 antibodies of the invention have following at least two or at least 3,4,5,6,7,8,9 or 10 kind Any combinations of property.In another aspect, humanization anti-il-23 p 19 antibodies of the invention have following all properties.

To mankind IL-23 K_D≤ 1pM (association rate is unchanged in 50% human serum)

In vitro IL-23 is blocked to be combined with mankind IL-23R/Fc

Do not combined with human IL-12

ADCC/CDC activity without prediction

To stump-tailed macaque IL-23 K_D≤1pM

With mouse or rat IL-23 no cross reactions

Solubility >=100mg/ml (is such as measured by ultraviolet spectroscopy and monitored by turbidity).

In another aspect, humanized antibody of the invention has at least one below in conjunction with property (property A).Another In aspect, humanization anti-il-23 p 19 antibodies of the invention have any combinations of following at least two or at least three kinds property. In another aspect, humanization anti-il-23 p 19 antibodies of the invention have following all properties.

To mankind IL-23 K_D≤ 1pM (association rate is unchanged in 50% human serum)

Do not combined with human IL-12

To stump-tailed macaque IL-23 K_D≤1pM

With mouse or rat IL-23 no cross reactions.

Particularly, the K of humanized antibody of the invention to mankind IL-23_D≤ 1pM (combines speed in 50% human serum Rate is unchanged) and do not combined with human IL-12.

In another aspect, humanized antibody of the invention has at least one following functions property (property B).Another In aspect, humanization anti-il-23 p 19 antibodies of the invention have any combinations of following at least two or at least three kinds property. In another aspect, humanization anti-il-23 p 19 antibodies of the invention have following all properties.

In vitro IL-23 is blocked to be combined with mankind IL-23R/Fc

Suppress to be produced (to two kinds of cells under 1mg/kg by the mankind IL-23 IL-17 induced and IL-22 in mouse ear The suppression of the factor is >=80%).

In another aspect, humanized antibody of the invention has at least one following property (property C).On the other hand In, humanization anti-il-23 p 19 antibodies of the invention have any combinations of following at least two or at least three kinds property.Another In one side, humanization anti-il-23 p 19 antibodies of the invention have following all properties.

ADCC/CDC activity without prediction

In another aspect, humanized antibody of the invention has at least one following property (property C).On the other hand In, humanization anti-il-23 p 19 antibodies of the invention have any combinations of following at least two property.In another aspect, originally The humanization anti-il-23 p 19 antibodies of invention have following all properties.

ADCC/CDC activity without prediction

In another aspect, humanized antibody of the invention has at least one property A, at least one property B and at least A kind of property C.In another aspect, humanization anti-il-23 p 19 antibodies of the invention have at least two or at least three kinds of properties A, B and C any combinations.

In certain aspects, humanized antibody shows blocking activity, and thus it subtracts the combination of IL-23 and DCRS5 It is small by least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, extremely Few 85%, at least 90% or at least 95%.Competitive binding analysis as known in the art can be used to measure antibody blocking IL- 23 abilities combined with DCRS5.Or can be by assessing IL-23 biological effect (such as IL-17 and IL-22 production It is raw) it is whether suppressed by the signal transduction of DCRS5 mediation to determine, the blocking activity of antibody is measured with this.

In another aspect, the present invention provides a kind of humanization anti-IL-23 p 19 with favourable biophysical properties and resisted Body.In an aspect, humanization anti-il-23 p 19 antibodies of the invention are with least 90% monomeric form or at least 92% monomer Form or at least 95% monomeric form is present in buffer solution.In another aspect, humanization anti-IL-23 p 19 of the invention resists At least 90% monomeric form or at least 92% monomeric form are kept in buffer solution for body or at least 95% monomeric form continues one The moon or four months.

In an aspect, humanized antibody of the invention is antibody A, antibody B, antibody C or antibody D.Therefore, at one In embodiment, humanized antibody of the invention includes sequence of light chain SEQ ID NO:174 and sequence of heavy chain SEQ ID NO:176 (antibody A).In another embodiment, humanized antibody of the invention includes sequence of light chain SEQ ID NO:174 and heavy chain Sequence SEQ ID NO:178 (antibody B).In another embodiment, humanized antibody of the invention includes sequence of light chain SEQ ID NO:180 and sequence of heavy chain SEQ ID NO:176 (antibody C).In another embodiment, humanized antibody of the invention Include sequence of light chain SEQ ID NO:180 and sequence of heavy chain SEQ ID NO:178 (antibody D).

In another embodiment, humanized antibody of the invention is by sequence of light chain SEQ ID NO:174 and heavy chain sequence Arrange SEQ ID NO:176 compositions (antibody A).In another embodiment, humanized antibody of the invention is by sequence of light chain SEQ ID NO:174 and sequence of heavy chain SEQ ID NO:178 compositions (antibody B).In another embodiment, humanization of the invention Antibody is by sequence of light chain SEQ ID NO:180 and sequence of heavy chain SEQ ID NO:176 compositions (antibody C).In another embodiment party In case, humanized antibody of the invention is by sequence of light chain SEQ ID NO:180 and sequence of heavy chain SEQ ID NO:178 compositions are (anti- Body D).

In some embodiments, humanization anti-il-23 p 19 antibodies (including its antigen-binding fragment, such as heavy chain and light Chain variable region) include have come from antibody A (sequence of light chain=SEQ ID NO:174；Sequence of heavy chain=SEQ ID NO:176)、 Antibody B (sequence of light chain=SEQ ID NO:174；Sequence of heavy chain=SEQ ID NO:178), antibody C (sequence of light chain=SEQ ID NO:180；Sequence of heavy chain=SEQ ID NO:Or antibody D (sequence of light chain=SEQ ID NO 176):180；Sequence of heavy chain=SEQ ID NO:178) amino acid sequence of residue.

In another embodiment, the present invention provides one kind by SEQ ID NO:181 amino acid residue 108 to 126 and amino acid residue 137 to 151 form epitope at the mankind IL-23p19 anti-il-23 p 19 antibodies combined or its antigen Binding fragment.

In another embodiment, the present invention provide it is a kind of with antibody of the invention (such as antibody A as described herein, Antibody B, antibody C or antibody D) competitive binding mankind IL-23p19 anti-il-23 p 19 antibodies or its antigen-binding fragment.Can Antibody or antigen-binding fragment competitive binding IL-23p19 are measured using competitive binding analysis as known in the art Ability.

Humanization anti-il-23 p 19 antibodies optionally include specific amino acids substitution in shared or germline skeleton area.Compared to By the way that CDR or HVL " directly exchanging (direct swap) " to the humanized antibody formed in human germline's skeleton area is shown The antibody efficiency shown, in foregoing frame position the specific substitution of amino acid residue can improve antibody efficiency many aspects (including Binding affinity and/or stability).

In some embodiments, the present invention illustrates that light chain variable district has SEQ ID NO:84、86、88、90、91、93、 95th, other monoclonals of amino acid sequence shown in 97,99,101,103,105,107,109,111,113,115,117 or 119 resist Body.In some embodiments, the present invention illustrates that weight chain variable district has SEQ ID NO:121、123、125、127、128、 130th, amino acid sequence shown in 132,134,136,138,140,142,144,146,148,150,152,154 or 156 is other Monoclonal antibody (referring to upper table 1 and table 2).The CDR sequence of these mouse antibodies is showed in table 3 and table 4.By these CDR It is placed in the mankind and shares in the FR of heavy chain and light-chain variable domain and will produce applicable humanized antibody of the invention.

Particularly, what the present invention provided light chain variable district and weight chain variable district is combined as SEQ ID NO:84/121、86/ 123、88/125、90/127、91/128、93/130、95/132、97/134、99/136、101/138、103/140、105/142、 107/144th, 109/146,111/148,113/150,115/152,117/154 or 119/156 monoclonal antibody.These are variable Area can combine with human constant regions.

In some embodiments, the present invention illustrates that light-chain variable sequence has SEQ ID NO:158th, 160,162 or Other humanized antibodies of amino acid sequence shown in 164.In some embodiments, the present invention illustrates weight chain variabl area sequence tool There are SEQ ID NO:166th, other humanized antibodies of amino acid sequence shown in 168,170 or 172 (referring to upper table 5 and table 6). The CDR sequence of these antibody is showed in table 3 and table 4.Particularly, the present invention provides the group of light chain variable district and weight chain variable district It is combined into SEQ ID NO:160/166th, 160/168,158/166 or 158/168 monoclonal antibody.These variable regions can be with the mankind Constant region combines.

In another embodiment, the present invention relates to a kind of anti-il-23 p 19 antibodies or its antigen-binding fragment, it is wrapped Contain：Humanization light-chain variable domain, the light-chain variable domain include SEQ ID NO:160 CDR, and amino acid sequence and SEQ ID NO:The skeleton region amino acid sequence at least 90% of 160 variable domain light-chain amino acid sequence is consistent, at least 93% unanimously or at least 95% consistent skeleton area；And humanized heavy chain's variable domain, the heavy chain variable domain include SEQ ID NO:166 CDR, and ammonia Base acid sequence and SEQ ID NO:The skeleton region amino acid sequence at least 90% of 166 variable domain heavy chain amino acid sequence is consistent, At least 93% consistent or at least 95% consistent skeleton area.In one embodiment, anti-il-23 p 19 antibodies are humanization list Clonal antibody.

In another embodiment, the present invention relates to a kind of anti-il-23 p 19 antibodies or its antigen-binding fragment, it is wrapped Contain：Humanization light-chain variable domain, the light-chain variable domain include SEQ ID NO:160 CDR, and amino acid sequence and SEQ ID NO:The skeleton region amino acid sequence at least 90% of 160 variable domain light-chain amino acid sequence is consistent, at least 93% unanimously or at least 95% consistent skeleton area；And humanized heavy chain's variable domain, the heavy chain variable domain include SEQ ID NO:168 CDR, and ammonia Base acid sequence and SEQ ID NO:The skeleton region amino acid sequence at least 90% of 168 variable domain heavy chain amino acid sequence is consistent, At least 93% consistent or at least 95% consistent skeleton area.In one embodiment, anti-il-23 p 19 antibodies are humanization list Clonal antibody.

In another embodiment, the present invention relates to a kind of anti-il-23 p 19 antibodies or its antigen-binding fragment, it is wrapped Contain：Humanization light-chain variable domain, the light-chain variable domain include SEQ ID NO:158 CDR, and amino acid sequence and SEQ ID NO:The skeleton region amino acid sequence at least 90% of 158 variable domain light-chain amino acid sequence is consistent, at least 93% unanimously or at least 95% consistent skeleton area；And humanized heavy chain's variable domain, the heavy chain variable domain include SEQ ID NO:166 CDR, and ammonia Base acid sequence and SEQ ID NO:The skeleton region amino acid sequence at least 90% of 166 variable domain heavy chain amino acid sequence is consistent, At least 93% consistent or at least 95% consistent skeleton area.In one embodiment, anti-il-23 p 19 antibodies are humanization list Clonal antibody.

In another embodiment, the present invention relates to a kind of anti-il-23 p 19 antibodies or its antigen-binding fragment, it is wrapped Contain：Humanization light-chain variable domain, the light-chain variable domain include SEQ ID NO:158 CDR, and amino acid sequence and SEQ ID NO:The skeleton region amino acid sequence at least 90% of 158 variable domain light-chain amino acid sequence is consistent, at least 93% unanimously or at least 95% consistent skeleton area；And humanized heavy chain's variable domain, the heavy chain variable domain include SEQ ID NO:168 CDR, and ammonia Base acid sequence and SEQ ID NO:The skeleton region amino acid sequence at least 90% of 168 variable domain heavy chain amino acid sequence is consistent, At least 93% consistent or at least 95% consistent skeleton area.In one embodiment, anti-il-23 p 19 antibodies are humanization list Clonal antibody.

In some specific embodiments, humanization anti-il-23 p 19 antibodies disclosed herein include at least heavy chain or light chain Variable domain, the light-chain variable domain include the CDR of the murine monoclonal antibody or humanized antibody as shown in upper table 1 to table 6 Or the HVL and FR of human germline's heavy chain and light-chain variable domain.

The CDR of these sequences is showed in table 3 and table 4.Therefore, in an aspect, the present invention provides a kind of anti-IL- 23p19 antibody or its antigen-binding fragment, it includes light chain CDR1 (L-CDR1) sequence SEQ ID NO:1、4、6、7、8、11、 15th, 18,19,22,27 or 30；Light chain CDR2 (L-CDR2) sequence SEQ ID NO:2nd, 5,9,12,16,20,23,25,28 or 31； Light chain CDR3 (L-CDR3) sequence SEQ ID NO:3rd, 10,13,14,17,21,24,26,29 or 32；Heavy chain CDR1 (H-CDR1) Sequence SEQ ID NO:33rd, 36,38,40,43,45,48,51,54,57,60,63,66,67,68,69,77 or 80；Heavy chain CDR2 (H-CDR2) sequence SEQ ID NO:34th, 39,41,46,49,52,55,58,61,64,70,72,73,75,78 or 81；And heavy chain CDR3 (H-CDR3) sequence SEQ ID NO:35th, 37,42,44,47,50,53,56,59,62,65,71,74,76,79 or 82. In one side, anti-il-23 p 19 antibodies or its antigen-binding fragment include：Light chain variable district, more than the light chain variable district includes Listed L-CDR1, L-CDR2 listed above and L-CDR3 listed above；And weight chain variable district, the weight chain variable district include above institute Arrange H-CDR1, H-CDR2 listed above and H-CDR3 listed above.

In another aspect, the present invention provides a kind of anti-il-23 p 19 antibodies or its antigen-binding fragment, and it is included：

A) it is respectively SEQ ID NO:1st, 2,3,33,34 and 35 L-CDR1, L-CDR2, L-CDR3, H-CDR1, H-CDR2 And H-CDR3 sequences；Or

B) it is respectively SEQ ID NO:4th, 5,3,36,34 and 37 L-CDR1, L-CDR2, L-CDR3, H-CDR1, H-CDR2 And H-CDR3 sequences；Or

C) it is respectively SEQ ID NO:1st, 2,3,38,39 and 35 L-CDR1, L-CDR2, L-CDR3, H-CDR1, H-CDR2 And H-CDR3 sequences；Or

D) it is respectively SEQ ID NO:6th, 2,3,40,41 and 42 L-CDR1, L-CDR2, L-CDR3, H-CDR1, H-CDR2 And H-CDR3 sequences；Or

E) it is respectively SEQ ID NO:7th, 2,3,43,41 and 44 L-CDR1, L-CDR2, L-CDR3, H-CDR1, H-CDR2 And H-CDR3 sequences；Or

F) it is respectively SEQ ID NO:8th, 9,10,45,46 and 47 L-CDR1, L-CDR2, L-CDR3, H-CDR1, H- CDR2 and H-CDR3 sequences；Or

G) it is respectively SEQ ID NO:8th, 9,10,48,49 and 50 L-CDR1, L-CDR2, L-CDR3, H-CDR1, H- CDR2 and H-CDR3 sequences；Or

H) it is respectively SEQ ID NO:11st, 12,13,51,52 and 53 L-CDR1, L-CDR2, L-CDR3, H-CDR1, H- CDR2 and H-CDR3 sequences；Or

I) it is respectively SEQ ID NO:7th, 2,14,54,55 and 56 L-CDR1, L-CDR2, L-CDR3, H-CDR1, H- CDR2 and H-CDR3 sequences；Or

J) it is respectively SEQ ID NO:15th, 16,17,57,58 and 59 L-CDR1, L-CDR2, L-CDR3, H-CDR1, H- CDR2 and H-CDR3 sequences；Or

K) it is respectively SEQ ID NO:18th, 16,17,60,61 and 62 L-CDR1, L-CDR2, L-CDR3, H-CDR1, H- CDR2 and H-CDR3 sequences；Or

L) it is respectively SEQ ID NO:19；20；21；63rd, 66,67 or 68；64；And 65 L-CDR1, L-CDR2, L- CDR3, H-CDR1, H-CDR2 and H-CDR3 sequence；Or

M) it is respectively SEQ ID NO:22nd, 23,24,69,70 and 71 L-CDR1, L-CDR2, L-CDR3, H-CDR1, H- CDR2 and H-CDR3 sequences；Or

N) it is respectively SEQ ID NO:22nd, 25,26,55,72 and 71 L-CDR1, L-CDR2, L-CDR3, H-CDR1, H- CDR2 and H-CDR3 sequences；Or

O) it is respectively SEQ ID NO:8th, 9,10,45,73 and 74 L-CDR1, L-CDR2, L-CDR3, H-CDR1, H- CDR2 and H-CDR3 sequences；Or

P) it is respectively SEQ ID NO:27th, 28,29,45,75 and 76 L-CDR1, L-CDR2, L-CDR3, H-CDR1, H- CDR2 and H-CDR3 sequences；Or

Q) it is respectively SEQ ID NO:8th, 9,10,77,78 and 79 L-CDR1, L-CDR2, L-CDR3, H-CDR1, H- CDR2 and H-CDR3 sequences；Or

R) it is respectively SEQ ID NO:30th, 31,32,80,81 and 82 L-CDR1, L-CDR2, L-CDR3, H-CDR1, H- CDR2 and H-CDR3 sequences.

In an aspect, anti-il-23 p 19 antibodies or its antigen-binding fragment include：Light chain variable district, the light chain variable Combined comprising L-CDR1, L-CDR2 and L-CDR3 listed above in area；And weight chain variable district, the weight chain variable district include listed above H-CDR1, H-CDR2 and H-CDR3 are combined.

In a particular embodiment, CDR region exchanges (that is, such as a kind of mouse between these exemplary immunoglobulins Antibody or come from one or two CDR of its humanized antibody with from another mouse antibodies or the humanized antibody for coming from it Similar CDR is exchanged) chimeric antibody be expected that applicable antibody can be produced.

In certain embodiments, humanization anti-il-23 P19 antibody is antibody fragment.Various antibody pieces are summarized above Section and have been developed that multiple technologies to produce the antibody fragment.Fragment can be obtained via proteolytic digestion complete antibody (for example, with reference to Morimoto et al., 1992, Journal of Biochemical and Biophysical Methods 24:107-117；And Brennan et al., 1985, Science 229:81).Or can directly it be produced in recombinant host cell Fragment.For example, Fab'-SH fragments can be reclaimed directly from Escherichia coli (E.coli), and implement chemical coupling to form F (ab')₂ Fragment (for example, with reference to Carter et al., 1992, Bio/Technology 10:163-167)., can be direct by another method F (ab') is separated from restructuring host cell cultures₂Fragment.Those skilled in the art can be appreciated that other of generation antibody fragment Technology.Therefore, in an aspect, the present invention provides antibody fragment, and it includes CDR as described herein, particularly described herein L-CDR1, L-CDR2, L-CDR3, H-CDR1, H-CDR2 and H-CDR3 a kind of combination.In another aspect, the present invention carries For antibody fragment, it includes variable region as described herein, such as one kind of light chain variable district as described herein and weight chain variable district Combination.

Some embodiments include including sequence of light chain SEQ ID NO:Any one of 174 or 180 and sequence of heavy chain SEQ ID NO:The F (ab') of the humanization anti-il-23 p 19 antibodies of 176 or 178 combinations₂Fragment.These embodiments may include to include The F (ab')₂Complete antibody.

In some embodiments, the antibody or antibody fragment include the constant region of mediation effector function.Constant region can Antibody-dependent cytotoxicity (ADCC), the antibody dependent cellular phagocytosis of target cell for expression IL-23 are provided (ADCP) and/or complement-dependent cytotoxicity (CDC) is reacted.Effector domain can be (such as) the Fc areas of Ig molecules.

The effector domain of antibody may be from any suitable invertebrate species and isotype.From the same of different animals species The ability of kind type mediation effector function is different.For example, human immunoglobulin(HIg) mediation CDC and ADCC/ADCP ability is typically divided Ju You not following sequence：IgM≈IgG₁≈IgG₃>IgG₂>IgG₄And IgG₁≈IgG₃>IgG₂/IgM/IgG₄.Muroid immune globulin White general mediation CDC and ADCC/ADCP typically has following sequence respectively：Muroid IgM ≈ IgG₃>>IgG_2b>IgG_2a>>IgG₁And IgG_2b>IgG_2a>IgG₁>>IgG₃.In another embodiment, muroid IgG_2aMediation ADCC, and muroid IgG_2aAnd mediation both IgM CDC。

Antibody modification

Humanization anti-il-23 P19 antibody and medicine may include to humanization anti-il-23 P19 antibody or its antigen binding fragment The modification of section.For example, it may be desirable to the modified antibodies in terms of effector function, to strengthen effect of the antibody in treating cancer.One It is modified to described in kind and cysteine residues is introduced into Fc areas, so that interchain disulfide bond can be formed in the region.Thus The same diabodies of generation can have the complement-mediated cell killing and/or antibody of improved internalization ability and/or enhancing Dependent cellular cytotoxicity (ADCC).For example, with reference to Caron et al., 1992, J.Exp Med.176:1191-1195；And Shopes, 1992, J.Immunol.148：2918-2922.Also the agent of isodigeranyl functional cross-links can be used has enhancing to prepare The same diabodies of antitumor activity, such as Wolff et al., 1993, Cancer Research 53：Described in 2560-2565.Or Person, antibody can be engineered with containing dual Fc areas, so as to strengthen the complement lysis of antibody and ADCC abilities.Referring to Stevenson Et al., 1989, Anti-Cancer Drug Design 3：219-230.

The antibody for supporting that ADCC ability is improved is generated by changing the glycosylation pattern of antibody Fc district.This is can Can, because antibody is in C_H2In domain glycosylation at asparagine residue N297 participate in necessary to ADCC IgG and Fc γ acceptors it Between interaction.Engineered host cell system with express glycosylation change (such as increase divide type (bisecting) N- second equally Acyl gucosamine or reduce fucose) antibody.The presence that reduction fucose divides type N-Acetyl-D-glucosamine equally than increase is bigger Strengthen ADCC activity to degree.In addition, the ADCC enhancings of low fucose antibody are unrelated with Fc γ RIIIa V/F polymorphisms.

The glycosylation engineered alternative that amino acid sequence in modified antibodies Fc areas is enhancing ADCC.By extensive Mutation analysis determine the Fc γ acceptors in human IgG₁On binding site.This causes humanization IgG of the generation with Fc mutation₁ Antibody, the mutation improve the binding affinity to Fc γ RIIIa and strengthen in vitro ADCC.In addition, binding characteristic is obtained Fc variants with a variety of different changes, the characteristic for example and specific Fc γ R acceptors combination enhancing and with other Fc γ R The combination of acceptor is constant or reduces.

On the other hand immunoconjugates are included, it includes the humanized antibody being conjugated with cytotoxic agent or its fragment, should Cytotoxic agent be such as chemotherapeutic agent, toxin (for example, bacterium, fungi, plant or animal origin enzyme activity toxin or Its fragment) or radio isotope (that is, radioactivity conjugate).

Have been described available for the chemotherapeutic agent for producing the immunoconjugates.Sew available for available be immunized is formed The enzymatic activity toxin and its fragment of compound include diphtheria A chains, the nonbinding active fragments of diphtheria toxin, exotoxin A chain and (come from Pseudomonas aeruginosa (Pseudomonas aeruginosa)), ricin A chain, abrin A chain, modeccin A Chain, α-sarcine, tung oil tree (Aleurites fordii) albumen, China pink fibroin, dyers' grapes (Phytolaca Americana) albumen (PAPI, PAPII and PAP-S), balsam pear (Momordica charantia) inhibitor, curcin (curcin), crotin, Saponaria officinalis (Sapaonaria officinalis) inhibitor, gelonin, mitogellin, Restrictocin, phenomycin, enomycin (enomycin), trichothecene etc..A variety of radionuclides are put available for generation The humanization anti-il-23 P19 antibody of penetrating property conjugate.Example includes²¹²Bi、¹³¹I、¹³¹In、⁹⁰Y and¹⁸⁶Re。

Humanization anti-il-23 P19 antibody can be made with the conjugate of cytotoxic agent or chemotherapeutant by known method It is made with a variety of bifunctional protein coupling agents, the coupling agent such as 3- (2- pyridine radicals dimercapto) propionic acid N- succinimide esters (SPDP), imido grpup tiacyclopentane (IT), the dual-function derivative (such as dimethyl adipate HCL) of imidoether, activity Ester (such as disuccinimidyl suberate), aldehyde (such as glutaraldehyde), two-fold nitrilo compound (such as double (p- triazobenzenes Formoxyl) hexamethylene diamine), dual azepine derivatives (such as double (to diazoniumbenzoyl)-ethylenediamines), diisocyanate (such as first Benzene 2,6- diisocyanate) and double activated fluorine compounds (such as the fluoro- 2,4- dinitro benzenes of 1,5- bis-).For example, ricin (WA) is exempted from Epidemic disease toxin can such as Vitetta et al., 1987, Science 238:Prepared described in 1098.The different sulphur cyanogen of 1- marked through carbon-14 Acyl benzyl -3- methyl diethylene-triamine pentaacetic acid (MX-DTPA) is the exemplary chelating of radioactive nucleotides and antibody conjugate Agent.Also cleavable joint can be used to be formed for conjugate.

Also humanization anti-il-23 P19 antibody disclosed herein can be allocated as immunoliposome.Liposome containing antibody Prepared by methods known in the art, such as illustrated in documents below：Epstein et al., 1985, Proc.Natl.Acad.Sci.USA 82:3688；Hwang et al., 1980, Proc.Natl.Acad.Sci.USA 77:4030； And U.S. Patent No. No. 4,485,045 and No. 4,544,545.The liposome that circulation time extends be disclosed in (such as) U.S. is special In profit the 5,013,556th.

The liposome being particularly useful, which can be used, includes phosphatidyl choline, cholesterol and phosphatidyl-ethanolamine derived from PEG (PEG-PE) lipid composition is generated by reverse phase evaporation.Liposome is extruded via the filter membrane with specified aperture to obtain There must be the liposome of desired diameter.The Fab' fragments and lipid for making antibody disclosed herein can be reacted via disulfide exchange Body combines, such as Martin et al., 1982, J.Biol.Chem.257:Described in 286-288.Chemotherapeutic agent (such as it is how soft Than star) optionally it is contained in liposome.For example, with reference to Gabizon et al., 1989, J.National Cancer Inst.81 (19):1484。

Illustrate herein and disclosed antibody also can be used for ADEPT (antibody by the way that the enzyme of antibody and activated prodrugs is conjugated The enzyme prodrug therapy of guiding) operate, prodrug (for example, peptidyl chemotherapeutic agent) is converted into active anti-cancer drug by the enzyme.Example Such as, referring to WO 81/01145, WO 88/07378 and U.S. Patent No. 4,975,278.Immunoconjugates available for ADEPT In enzyme component for the enzyme that prodrug is translated into the stronger cytotoxic form of activity can be acted on.Available for ADEPT's Specific enzyme includes but is not limited to：Alkaline phosphatase, it is free drug that it, which is used for phosphate ester-containing pro-drug conversion,；Aromatic yl acid ester Enzyme, it is used to sulfur acid ester prodrugs being converted into free drug；Cytosine deaminase, it is used to turn non-toxic 5-flurocytosine Turn to cancer therapy drug 5 FU 5 fluorouracil；Protease, such as husky thunder bacterium (Serratia) protease, thermolysin, withered grass bar Mycoproteinase, carboxypeptidase and cathepsin (such as cathepsin B and L), it is free medicine that it, which is used for pro-drug conversion containing peptide, Thing；D- alanylcarboxypeptidases, it is used to convert the prodrug containing D- 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor bases；Carbohydrate-cleaving enzyme, such as β- Galactosidase and neuraminidase, it is used to glycosylated prodrugs being converted into free drug；Beta-lactamase, it is used to pass through Medicine is converted into free drug derived from beta-lactam；And penicillin amidase, such as Penicillin-V-Amidase or benzyl penicillin acyl Amine enzyme, it is used to that free drug will to be converted into through medicine derived from nitrophenoxyacetyl or phenyl acetyl on amine nitrogen respectively.Or Person, can be used the antibody (" abzyme ") with enzymatic activity by pro-drug conversion be free active medicine (for example, with reference to Massey, 1987, Nature 328：457-458).Antibody-antibody enzyme conjugate can be prepared for that will resist by known method Body enzyme is delivered to tumor cell group, for example, by making enzyme be tried with above-mentioned humanization anti-il-23 P19 antibody/isodigeranyl functional cross-links It is prepared by agent covalent bond.Or recombinant DNA technology construction fusion protein can be used, it includes antibody disclosed herein extremely Few antigen binding domain and at least functional activity part for the above-mentioned enzyme being attached thereto (for example, with reference to Neuberger et al., 1984, Nature 312:604-608)。

In certain embodiments, it may be desirable to using humanization anti-il-23 P19 antibody fragments rather than complete antibody with (example As) promotion organization infiltration.Modified antibodies fragment can it is expected to extend its serum half-life.This can by (such as) by salvage receptor Included with reference to epitope in antibody fragment to reach.In one approach, can change suitable in (such as being allowed to be mutated) antibody fragment Suitable region, or can by (such as) DNA or peptide symthesis include epitope in peptide tag, then make the label in end or at middle part Merged with antibody fragment.For example, with reference to WO 96/32478.

In other embodiments, the covalent modification of humanization anti-il-23 P19 antibody is also included.Covalent modification include pair The modification of following group：Cysteinyl residues, Histidyl residues, lysyl- and n terminal residue, arginyl- are residual Base, tyrosyl- residue, carboxyl side group (aspartyl or glutamyl), glutaminase acyl group and N acyl group are residual Base or seryl- or threonyl residues.Another covalent modification type, which is related to, makes glucosides and antibody chemistry or enzymatic of glucosides.If It is applicable, the modification can be prepared by chemical synthesis or by the enzymatic of antibody or chemical cracking.Can be by making the target of antibody Reacted to amino acid residue with the organic derivatizing agents that can be reacted with selected side chain or amino-or carboxyl-tenninus residue by antibody Other covalent modification types be introduced into molecule.

The removal of any carbohydrate portions present on antibody can be completed with chemistry or enzymatic.Chemistry is gone Glycosylation is set forth in documents below：Hakimuddin et al., 1987, Arch.Biochem.Biophys.259:52 and Edge Et al., 1981, Anal.Biochem., 118:131.The enzymatic lysis of carbohydrate portions can be by using a variety of on antibody Endoglycosidase and exoglycosidase are reached, such as Thotakura et al., 1987, Meth.Enzymol 138:Described in 350.

The another type of available covalent modification include make antibody and a variety of nonproteinaceous polymers (such as polyethylene glycol, Polypropylene glycol or polyoxyalkylene) one of connected in a manner of described in one or more of documents below：U.S. Patent No. 4, No. 640,835, U.S. Patent No. 4,496,689, U.S. Patent No. 4,301,144, U.S. Patent No. 4,670,417, U.S. Patent No. 4,791,192 and U.S. Patent No. 4,179,337.

Humanization and amino acid sequence variation

The amino acid sequence variation of anti-il-23 P19 antibody can be resisted by the way that Suitable nucleotide is changed into introducing anti-il-23 P19 Prepared in body DNA or by peptide symthesis.The variant include (such as) embodiment hereof moderate resistance IL-23P19 antibody amino acids The missing of residue and/or insertion and/or substitution in sequence.Implement any combination of missing, insertion and substitution to obtain final structure Body is built, condition is that final construct has desired character.Amino acid change can also change humanization or variant anti-il-23 P19 resists Process after the translation of body, such as change number or the position of glycosylation site.

It is referred to as " the third ammonia available for the preferably method in some residues of mutagenesis position or region in identification anti-il-23 P19 antibody Sour scanning mutagenesis ", as Cunningham and Wells (Science, 244:1081-1085 (1989)) described in.Herein, identify Residue or target residues group (such as charged residue, such as arg, asp, his, lys and glu) and its through neutral or negatively charged Lotus amino acid (being usually alanine) substitutes to influence the interaction of amino acid and IL-23P19 antigens.Then by substituting Site introduces other or other variants to improve those displays to substituting the amino with function sensitive for substitution site Sour position.Therefore, when predefining the site for introducing variant amino acid sequence, the property of mutation itself is without predefining. For example, to analyze to the performance being mutated at anchor point, implement alanine scanning mutagenesis or random at target codon or region Mutagenesis and the anti-il-23 P19 antibody variants expressed by for expectation screening active ingredients.

Amino acid sequence insertion include amino-and/or carboxyl-terminal fusion (length between a residue to contain it is up to a hundred or In the range of the polypeptide of more residues) and the sequence of single or multiple amino acid residue in insert.The example bag of end insertion Include the anti-il-23 P19 antibody merged with epitope tag.Other insertion property variants of anti-il-23 P19 antibody molecules include enzyme or can Improve the polypeptide of antibody serum half-life period and merging for anti-il-23 P19 antibody N- or C- ends.

Another type of change system amino acid substitution variant.The variant removes at least in anti-il-23 P19 antibody molecules One amino acid residue simultaneously inserts different residues in the opening position.For substituting the site of greatest concern of mutagenesis to include hypermutation Area, but also cover FR changes.Conservative replacement is showed under " preferably substituting " title of table 5.If the substitution causes bioactivity Change, then can introduce prior change and (be named as " exemplary substitution ", or further explained referring below to amino acid classes State), and screen product.

Table 8：

In protein chemistry, the biological characteristics of generally accepted antibody can be by selecting to maintain the effect of following characteristic notable Different substitution is reached：(a) structure of polypeptide backbone in region is substituted, such as in folding or helical conformation, (b) molecule is in target Electric charge or hydrophobicity at site, or (c) side-chain bulk.Natural residue is divided into according to common side chain properties following all kinds of：

(1) hydrophobicity：Nor-leucine, met, ala, val, leu, ile；

(2) Neutral hydrophilic：cys、ser、thr；

(3) it is acid：asp、glu；

(4) it is alkaline：asn、gin、his、lys、arg；

(5) residue of chain orientation is influenceed：gly、pro；And

(6) aromatic series：trp、tyr、phe.

Non-conservation, which substitutes, to need the member of one of the species being exchanged for another species.

When also may replace and (typically substituted with serine) the appropriate conformation for maintaining humanization or variant anti-il-23 P19 antibody Any cysteine residues being not directed to, so as to put forward high molecular oxidation stability, prevent abnormal crosslinking, or offer and cell toxicant Property or cytostatic compound have confirmed that through chalaza.On the contrary, cysteine key can be improved its stabilization added to antibody Property (especially when antibody is the antibody fragment such as Fv fragments).

A kind of substituted type variant is related to one or more hypervariable regions in substitution parental generation antibody (such as humanization or human antibody) Residue.Generally, the selected gained variant for further researching and developing can have improvement relative to the parental generation antibody for producing the variant Biological nature.The convenient method for producing the substituted type variant is the affine sexal maturity implemented using phage display.Letter speech It, makes some hypervariable region sites (such as 6 to 7 sites) mutation produce all possible 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor in each site. Resulting antibody variants by monovalent fashion in the form of with the merging of M13 gene III products packaged in each particle from Filamentous phage particle is shown.Then the change of phage display is screened for biological activity (such as binding affinity) Body.To determine the candidate hypervariable region site of modification, alanine scanning mutagenesis can be implemented to determine to remarkably promote the super of antigen binding Become area's residue.Besides or furthermore, it is advantageous that the crystal structure of antigen-antibody complex is analyzed to determine antibody and people IL- Contact point between 23P19.The contact residues and neighbouring residue substitute residue for the candidate of technology described herein.Described in generation After variant, variant group is screened as described herein immediately, and may be selected that there is excellent spy in one or more correlation analyses Property antibody for further research and development.

The another kind of amino acid variant of antibody changes the original glycosylation pattern of antibody." change " means to lack one or more It is present in the carbohydrate portions in antibody and/or adds the glycosylation site that is not present in one or more antibody.

In some embodiments, it may be desirable to modify antibody of the invention to add glycosylation site.The glycosylation of antibody Usually N- connections or O- connections.N- connections refer to that carbohydrate portions are connected to ASPARTIC ACID residue side chains.Tripeptides sequence Row ASPARTIC ACID-X- serines and ASPARTIC ACID-X- threonines (wherein X is any amino acid in addition to proline) are carbon Carbohydrate moiety is connected to the recognition sequence of ASPARTIC ACID side chain through enzyme.Therefore, exist in polypeptide in the tripeptide sequence Any one can produce potential glycosylation site.O- connections glycosylation refers to sugared N- acetylgalactosamines, galactolipin or xylose One of be connected to hydroxy-amino-acid, most commonly serine or threonine, but 5-OxoPro or 5- hydroxylysines also can be used. Therefore, it is glycosylation specific protein (such as antibody), transforms the amino acid sequence of the albumen with containing in above-mentioned tripeptide sequence One or more (for N- connections glycosylation site).Also it can add or be replaced in by one or more serines or threonine residues Change in original antibodies sequence (for O- connections glycosylation site).

The nucleic acid molecules for encoding anti-il-23 P19 antibody amino acids sequence variants are by a variety of method systems known in the art It is standby.Methods described is including but not limited to from natural origin separation (in the case of natural acid sequence variant) or by anti- The previously prepared variant of IL-23P19 antibody or non-variant form implement oligonucleotide mediated (or fixed point) mutagenesis, PCR mutagenesis And prepared by cassette mutagenesis.

Polynucleotide, carrier, host cell and recombination method

Other embodiments cover the polynucleotide of the separation of the sequence comprising encoding humanized anti-il-23 P19 antibody, bag Carrier and host cell containing the polynucleotide, and produce the recombinant technique of humanized antibody.The polynucleotide of separation can be compiled Any desired form of code anti-il-23 P19 antibody, including such as full length monoclonal antibodies, Fab, Fab', F (ab')₂And Fv pieces Section, bivalent antibody (diabodies), linear antibodies, single-chain antibody molecules and the multi-specificity antibody formed from antibody fragment.

Some embodiments include the polynucleotide of separation, and it includes the light chain variable district of encoding antibody or antibody fragment Sequence, the light chain variable district have following any amino acid sequence：SEQ ID NO:84、86、88、90、91、93、95、97、99、 101st, 103,105,107,109,111,113,115,117 or 119.Encode the exemplary polynucleotide sequence of these amino acid sequences It is classified as SEQ ID NO:83rd, 85,87,89,92,94,96,98,100,102,104,106,108,110,112,114,116 and 118.Some embodiments include the polynucleotide of separation, and it includes the sequence of the weight chain variable district of encoding antibody or antibody fragment Row, the weight chain variable district have amino acid sequence SEQ ID NO:121、123、125、127、128、130、132、134、136、 138th, 140,142,144,146,148,150,152,154 or 156.Encode the exemplary polynucleotide sequence of these amino acid sequences It is classified as SEQ ID NO:120、122、124、126、129、131、133、135、137、139、141、143、145、147、149、 151st, 153 or 155.

Some embodiments include the polynucleotide of separation, and it includes the light chain variable district of encoding antibody or antibody fragment Sequence, the light chain variable district have following any amino acid sequence：SEQ ID NO:158th, 160,162 or 164.Encode these ammonia The exemplary polynucleotide sequence of base acid sequence is SEQ ID NO:157th, 159,161 or 163.Some embodiments include separation Polynucleotide, it includes the sequence of the weight chain variable district of encoding antibody or antibody fragment, and the weight chain variable district has amino acid Sequence SEQ ID NO:166th, 168,170 or 172.The exemplary polynucleotide sequence for encoding these amino acid sequences is SEQ ID NO:165th, 167,169 or 171.

Some embodiments include separation polynucleotide, it include the sequence of encoding antibody light, the light chain with Under any amino acid sequence：SEQ ID NO:174 or 180.The exemplary polynucleotide sequence for encoding these amino acid sequences is SEQ ID NO:173 or 179.Some embodiments include the polynucleotide of separation, and it includes the sequence of encoding antibody heavy, should Heavy chain has amino acid sequence SEQ ID NO:176 or 178.The exemplary polynucleotide sequence for encoding these amino acid sequences is SEQ ID NO:175 or 177.

In an aspect, the polynucleotide sequence encoding antibody or antibody fragment of separation, the antibody or antibody fragment The light chain variable district and weight chain variable district of following amino acid sequence are included respectively：SEQ ID NO:174 and SEQ ID NO:176； SEQ ID NO:174 and SEQ ID NO:178；SEQ ID NO:180 and SEQ ID NO:176；SEQ ID NO:180 and SEQ ID NO:178.The exemplary polynucleotide sequence for encoding these amino acid sequences is respectively SEQ ID NO:173 and 175；SEQ ID NO:173 and 177；SEQ ID NO:179 and 175；SEQ ID NO:179 and 177.

Can be with one or more comprising encoding humanized anti-il-23 P19 antibody or the polynucleotide of its fragment or the sequence of chain Regulation known in the art or control sequence fusion, and suitable expression vector or host cell known in the art can be contained in In.Each polynucleotide molecule of encoding heavy chain or light-chain variable domain all can be independently with encoding constant domain (such as people's constant domain) Polynucleotide sequence fusion so that complete antibody can be produced.Or polynucleotide or part thereof fusion can be made one Rise, so as to provide the template for producing single-chain antibody.

Produced for restructuring, by the polynucleotide of encoding antibody insertion replicable vector for clone's (DNA amplification) or Expression.The carrier of a variety of suitable expressing recombinant antibodies can be used.Carrier component generally comprise below (but not limited to) in one or More persons：Signal sequence, replication orgin, one or more marker gene, enhancing sub-component, promoter and transcription terminator.

Humanization anti-il-23 P19 antibody can also fused polypeptide form produce, wherein antibody and heterologous polypeptide, example There are other polypeptides of specific cleavage site such as signal sequence or in the amino terminal of maturation protein or polypeptide.Selected heterologous Signal sequence is usually the sequence that host cell can recognize that and processed (that is, being cracked by signal peptidase).For that can not identify simultaneously Process for the prokaryotic host cell of humanization anti-il-23 P19 antibody signal sequences, signal sequence can be taken by prokaryotic signal sequence Generation.Signal sequence can be (such as) alkaline phosphatase, penicillase, lipoprotein, heat-stable toxin II targeting sequencings etc..For ferment Mother stock is secreted, can with (such as) signals below substitutes signal sequences native：Targeting sequencing (bag derived from yeast invertase α-factor Include Blastocystis (Saccharomyces) and Kluyveromyces (Kluyveromyces) α-factor leaders), acid phosphorus Signal described in sour enzyme, Candida albicans (C.albicans) glucoamylase or WO90/13646.In mammalian cell In, mammalian signal sequences and viral secretory leaders (such as herpes simplex gD signal) can be used.The precursor DNAs of the DNA in area with encoding humanized anti-il-23 P19 antibody in reading frame is engaged.

Expression and cloning vector contain the nucleotide sequence for enabling carrier to be replicated in one or more selected host cells.It is logical Often, in cloning vector, this sequence for enable carrier independently of host chromosome DNA duplicator, and it includes replication orgin Or autonomously replicating sequence.The sequence for various bacteria, yeast and virus is known in the art.From pBR322 plasmid Replication orgin be applied to most of grams (Gram) negative bacteria, 2- υ plasmid origins are applied to yeast, and a variety of viruses rise Point (SV40, polyoma, adenovirus, VSV and BPV) can be used for the cloning vector in mammalian cell.Generally, mammal is expressed Carrier does not need replication orgin component (SV40 starting points can generally be used only, because it contains early promoter).

The gene of expression and cloning vector containing encoding selectable markers thing is to contribute to the identification to expression.Typical case can Selectable marker gene encodes following albumen：Assign to antibiotic or other toxin (for example, ampicillin (ampicillin), new Mycin, methotrexate (MTX) or tetracycline) resistance；Or the complement for auxotrophy；Or supplied in other alternatives compound The specific nutrition element being not present in culture medium, such as the gene of the D-alanine racemase of coding bacillus (Bacilli).

One embodiment of selection scheme prevents the growth of host cell using medicine.Those successfully turn through heterologous gene The cell of change, which produces, assigns drug-fast albumen, and therefore can be survived in the selection scheme.The embodiment of the dominant selection makes With drug neomycin, mycophenolic acid and hygromycin.The conventional selectable marker of mammalian cell makes it possible to identification for those can Absorb the label of the cell of the nucleic acid of encoding humanized anti-il-23 P19 antibody, such as DHFR (dihyrofolate reductase), chest Glycosides kinases, metallothionein-I and-II (such as primate metallothionein gene), adenosine deaminase, ornithine decarboxylation Enzyme etc..First by cultivating all transformant in the culture medium containing methotrexate (MTX) (Mtx) (DHFR competitive antagonist) To identify the cell converted through DHFR Select genes.When using wild type DHFR, suitable host cell is that DHFR activity lacks Sunken Chinese hamster ovary (CHO) cell line (such as DG44).

Or encoded anti-il-23 P19 antibody, wild type DHFR protein and another selectable marker (such as amino Portugal Polyglycoside 3'- phosphotransferases (APH)) DNA sequence dna conversion or cotransformation host cell (especially containing endogenous DHFR Wild-type host) can by containing for the selectable marker selective agent (such as aminoglycoside antibiotics, such as Kanamycins (kanamycin), neomycin or G418) culture medium in make cell growth to be selected.For example, with reference to the U.S. Patent the 4,965,199th.

If implementing restructuring using yeast cells as host cell to produce, then it can be used and be present in yeast plasmid YRp7 In TRP1 genes (Stinchcomb et al., 1979, Nature 282：39) it is used as selectable marker.TRP1 genes are scarce The yeast mutants bacterial strain (for example, ATCC the 44076th or PEP4-1) of few ability grown in tryptophan provides selection mark Note thing (Jones, 1977, Genetics 85:12).The presence that then trp1 is damaged in yeast host cell genome can be provided with Environment is imitated to detect conversion by being grown when in the absence of tryptophan.Similarly, Leu2p defective yeasts bacterial strain (such as ATCC 20,622 or 38,626) by being supplied with the known plasmid of LEU2 genes.

In addition, the carrier derived from 1.6 μm of circular plasmids pKD1 can be used to convert kluyveromyces.Or use is reported It can be used for Kluyveromyces lactis (K.lactis.) (Van den in the expression system for producing recombinant bovine renin on a large scale Berg, 1990, Bio/Technology 8:135).Also the disclosed ripe weight of industrial strain secretion by Kluyveromyces Group human serum albumins stable multicopy expression vector (Fleer et al., 1991, Bio/Technology 9:968-975).

Expression and cloning vector usually contain HOST ORGANISMS and can recognize that and with encoding anti-il-23 P19 antibody or its polypeptide The promoter that the nucleic acid molecules of chain are operatively connected.Include phoA promoters, beta-lactam suitable for the promoter of prokaryotic hosts (such as tac starts for enzyme and lactose promoter system, alkaline phosphatase, tryptophan (trp) promoter systems and hybrid promoter Son).Other known promoters are also suitable.Promoter for bacterial system can also contain and encoding humanized anti-IL- Shine-Dalgamo (S.D.) sequence that the DNA of 23P19 antibody is operatively connected.

Known a variety of eukaryotic promoter sequences.In fact, all eukaryotic genes all have rich AT areas, it is located at transcription initiation At about 25 to 30 bases of site upstream.Another sequence found at the base of transcripting start point upstream 70 to 80 of many genes CNCAAT areas are classified as, wherein N can be any nucleotides.It is AATAAA sequences at the 3' ends of most of eukaryotic genes, it may The signal of poly- A tails is added to the 3' ends of coded sequence.All sequences are all inserted in carrier for expression of eukaryon with suitable way.

Include the promoter of following enzyme suitable for the example of the initiating sequence of yeast host：3-phosphoglycerate kinases or Other glycolytic ferments, such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructose Kinases, GPI, 3-phoshoglyceric acid mutase, pyruvate kinase, phosphotriose isomerase, phosphoric acid Portugal Grape sugar isomerase and glucokinase.

Another advantage of inducible promoter is to control transcription by growth conditions.The promoter includes following enzyme Yeast promoter area：Alcohol dehydrogenase 2, different cell pigment C, acid phosphatase, the derivative enzyme related to nitrogen metabolism, metallothionein In vain, glyceraldehyde-3-phosphate dehydrogenase and the enzyme of responsible maltose and galactose utilization.For Yeast expression appropriate carrier and open Mover is expanded on further in EP 73,657.Yeast enhancers advantageously can be also used together with Yeast promoter.

In mammalian host cell from carrier transcription humanization anti-il-23 P19 antibody by (such as) from following acquisition Promoter control：Viral genome, such as polyoma, bird pox virus, adenovirus (such as adenovirus 2), bovine papilloma virus, fowl Sarcoma virus, cytomegalovirus, retrovirus, hepatitis b virus and simian virus 40 (SV40)；Heterologous mammalian opens Mover, such as actin promoter or immunoglobulin promoter；Heat-shock promoters, condition are the promoter and host Cell system is compatible.

Easily to be obtained in the form of SV40 restriction fragments, it also contains for the early stage of SV40 viruses and late promoter SV40 virus origin of replication.The immediate early promoter of people's cell hugeization virus is easily with HindIII E restriction fragments Form obtains.The system for expressing DNA in mammalian hosts using bovine papilloma virus as carrier is disclosed in United States Patent (USP) In No. 4,419,446.The modification of this system is set forth in U.S. Patent No. 4,601,978.Also reference can be made to Reyes etc. People, 1982, Nature297:598-601, it is disclosed under the thymidine kinase promoter control from herpes simplex virus small People's p- interferon cDNA is expressed in mouse cell.Or Rous sarcoma virus (rous sarcoma virus) long end can be used Repetitive sequence is as promoter.

Another applicable components available for recombinant expression carrier are enhancer sequence, and it is used to improve in higher eucaryote The DNA of encoding humanized anti-il-23 P19 antibody transcription.Known a variety of enhancer sequences from mammalian genes (such as Globin, elastoser, albumin, α-fetoprotein and insulin).However, usually using the increasing from eukaryotic cell virus Hadron.SV40 enhancers (bp 100-270), the cytomegalovirus that example is included on the slow side of replication orgin (late side) are sick Malicious early promoter enhancer, polyoma enhancer and adenovirus cancers on the slow side of replication orgin.On activating eucaryon The description of the enhancing component of promoter also can be found in Yaniv, and 1982, Nature 297:17-18.Can be by enhancer montage to load 5' the or 3' opening positions of humanization anti-il-23 P19 antibody coding sequences in body, but it is preferably placed at the 5' sites of promoter.

For eukaryotic host cell (yeast, fungi, insect, plant, animal, people's cell or organic from other many cells The karyocyte of body) in expression vector also containing terminate transcription and stable mRNA needed for sequence.The sequence typically may be used From the 5' and its 3' non-translational region acquisition occasionally of eucaryote or viral DNA or cDNA.The region, which is contained, is encoding anti-IL- The nucleotide segment of polyadenylated fragments is transcribed into the mRNA of 23P19 antibody untranslated part.A kind of available transcription Termination component is bovine growth hormone polyadenylation area.Referring to WO94/11026 and its disclosed in expression vector.At some In embodiment, CHEF systems can be used to express for humanization anti-il-23 P19 antibody.(for example, with reference to U.S. Patent No. 5, 888, No. 809, the disclosure of which is incorporated herein by reference.)

The suitable host cell for cloning or expressing DNA in this paper carriers is above-mentioned prokaryotic, yeast cells or height Deng eukaryotic.Include eubacteria, such as Gram-negative or Gram-positive organism suitable for the prokaryotes of this purpose, Such as：Enterobacteriaceae (Enterobacteriaceae), such as Escherichia (Escherichia) (such as Escherichia coli), Enterobacter (Enterobacter), Erwinia (Erwinia), Klebsiella (Klebsiella), proteus Belong to (Proteus), Salmonella (Salmonella) (such as salmonella typhimurium (Salmonella Typhimurium)), Serratia (such as Serratia marcesens (Serratia marcescans)) and Shigella , and Bacillus (such as bacillus subtilis (B.subtilis) and bacillus licheniformis (Shigella) (B.licheniformis) (such as the bacillus licheniformis disclosed in the DD 266,710 published on April 12nd, 1989 41P)), pseudomonas (Pseudomonas) (such as pseudomonas aeruginosa) and streptomyces (Streptomyces).It is preferred that Escherichia coli cloning host be Escherichia coli 294 (ATCC 31,446), but other bacterial strains (such as Escherichia coli B, large intestine bar Bacterium X1776 (ATCC 31,537) and Escherichia coli W3110 (ATCC 27,325)) it is also suitable for.The embodiment to be exemplary and It is non-limiting.

In addition to prokaryotes, such as the eukaryotic microorganisms such as filamentous fungi or yeast are also compiled for humanization anti-il-23 P19 antibody The suitable clone of code carrier or expressive host.Saccharomyces cerevisiae (Saccharomyces cerevisiae) or common bread ferment Female (baker's yeast) is the most frequently used low eucaryon host microorganism.However, typically can be used various other genus and species and Bacterial strain and it is available in this article, such as Schizosaccharomyces (Schizosaccharomyces pombe)；Kluyveromyces place It is main, such as Kluyveromyces lactis, Kluyveromyces fragilis (K.fragilis) (ATCC 12,424), Bulgarian Crewe dimension Yeast (K.bulgaricus) (ATCC 16,045), Brunswick kluyveromyces (K.wickeramii) (ATCC 24,178), watt You are special kluyveromyces (K.waltii) (ATCC 56,500), drosophila kluyveromyces (K.drosophilarum) (ATCC 36,906), Kluyveromyces thermotolerans (K.thermotolerans) and equine this kluyveromyces (K.marxianus)；Ye Shi Saccharomyces (yarrowia) (EP 402,226)；Pichia Pastoris (Pichia pastoris) (EP 183,070)；Read Pearl Pseudomonas (Candida)；Li's Trichoderma (Trichoderma reesia) (EP244,234)；Neurospora crassa bacterium (Neurospora crassa)；Perhaps prosperous yeast is permitted in prosperous saccharomyces (Schwanniomyces), such as west (Schwanniomyces occidentalis)；And filamentous fungi, such as neurospora (Neurospora), Penicillium (Penicillium), Tolypocladium (Tolypocladium) and Eurotium (Aspergillus) host (such as aspergillus nidulans (A.nidulans) and aspergillus niger (A.niger)).

The Suitable host cells of expression glycosylation humanization anti-il-23 P19 antibody derive from multi-cell organism.Moved without vertebra The example of thing cell includes plant and insect cell, including (such as) a variety of baculoviral strains and variant and from for example following place The insect host cell of main corresponding license：Spodopterafrugiperda (Spodoptera frugiperda) (caterpillar), Aedes aegypti (Aedes aegypti) (mosquito), aedes albopictus (Aedes albopictus) (mosquito), Drosophila melanogaster (Drosophila Melanogaster) (drosophila) and silkworm (Bombyx mori) (silkworm).A variety of transfection Strain can obtain from open approach, Such as the Bm-5 strains of autographa california (Autographa californica) NPV L-1 variants and BmSNPV, and it is described Virus is used especially for transfecting bomyx mori cell.

Also the plant cell cultures of cotton, corn, potato, soybean, petunia, tomato and tobacco can be used to make For host.

In another aspect, humanization anti-il-23 P19 is expressed as implementing in vertebrate cells.Vertebrate is thin Breeding of the born of the same parents in (tissue cultures) are cultivated has turned into routine operation and technology is available anywhere.Mammalian host cell line can be used Example be through SV40 conversion monkey kidney CV1 for (COS-7, ATCC CRL 1651), human embryonic kidney cell line (293 systems or through be subcloned with For 293 cells, Graham et al. grown in the culture that suspends, 1977, J.Gen Virol.36：59), baby hamster kidney cell (BHK, ATCC CCL 10), Chinese hamster ovary cell/- DHFR1 (CHO, Urlaub et al., 1980, Proc.Natl.Acad.Sci.USA 77：4216；Such as DG44), Properties of Sertoli Cells Isolated from Mice Testis (TM4, Mather, 1980, Biol.Reprod.23:243-251), MK cells (CV1ATCC CCL 70), African green monkey kidney cell (VERO-76, ATCC CRL-1587), human cervical carcinoma cell (HELA, ATCC CCL 2), MDCK (MDCK, ATCC CCL 34), buffalo mouse liver are thin Born of the same parents (BRL 3A, ATCC CRL 1442), human pneumonocyte (W138, ATCC CCL 75), human liver cell (Hep G2, HB 8065), MMT (MMT 060562, ATCC CCL51), TR1 cells (Mather et al., 1982, Annals N.Y.Acad.Sci.383：44-68), the cells of MRC 5, FS4 cells and human hepatoma cell line (Hep G2).

It is used to producing expression or the cloning vector of humanization anti-il-23 P19 antibody with above-mentioned to convert host cell, and Cultivated in conventional nutrient medium, the nutrient medium is modified to be suitable for evoked promoter, selection transformant or amplification volume The gene of code expectation sequence.

The host cell for producing humanization anti-il-23 P19 antibody described herein can be cultivated in a variety of culture mediums.City Sell culture medium and be applied to culture host cell, such as Ham's F10 (Sigma-Aldrich companies, St.Louis, Mo.), minimum Dulbecco minimum essential medium Dulbecco ((MEM), Sigma-Aldrich companies), RPMI-1640 (Sigma-Aldrich companies) and Dulbecco Eagle culture medium (Dulbecco's Modified Eagle's Medium) ((DMEM), Sigma-Aldrich of improvement Company).In addition, any culture medium that one or more of documents below can be used to illustrate is used as the culture medium of host cell： Ham et al., 1979, Meth.Enz.58：44；Barnes et al., 1980, Anal.Biochem.102：255；U.S. Patent No. 4, No. 767,704, U.S. Patent No. 4,657,866, U.S. Patent No. 4,927,762, U.S. Patent No. 4,560,655, U.S. Patent No. 5,122,469, WO 90/103430 and WO 87/00195.Any one of described culture medium can be according to need Will be supplemented with hormone and/or other growth factors (such as insulin, transferrins or EGF), salt (such as chlorination Sodium, calcium salt, magnesium salts and phosphate), buffer solution (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotic (such as celebrate Big mycin), trace element (being defined as generally with inorganic compound existing for the ultimate density of micro-molar range) and glucose or The equivalent energy.Other supplements of debita spissitudo well known by persons skilled in the art can also be included.Such as the culture such as temperature, pH Condition is previously used for the condition of selected expression host cell for those, and is apparent to those skilled in the art 's.

When using recombinant technique, antibody can produce in the cell, in periplasmic space, or directly secrete to culture medium In.If producing antibody in the cell, as first step cell rupture can be made to discharge albumen.It can be moved by centrifugation or ultrafiltration Except particle debris, i.e. host cell or dissolving fragment.Carter et al., 1992, Bio/Technology 10:163-167 is illustrated Separation is secreted to the operation of the antibody in Escherichia coli periplasmic space.In short, in sodium acetate (pH 3.5), EDTA and benzene first sulphur Cell paste was set to thaw through about 30 minutes in the presence of acyl fluorides (PMSF).Cell fragment can be removed by centrifuging.If antibody-secreting Into culture medium, then generally first by (such as the Amicon or Millipore Pellicon ultrafiltration of commercial protein concentration filter Unit) supernatant of the concentration from the expression system.It may include the albumen enzyme level such as PMSF in any abovementioned steps Agent may include antibiotic to prevent external contaminant from growing to suppress proteolysis.A variety of methods can be used from host cell Separation antibody.

Can be used (such as) hydroxylapatite chromatography, gel electrophoresis, dialysis and affinity chromatography come be purified from cell preparation Antibody compositions, wherein affinity chromatography are conventional purification technique.Albumin A is anti-depending on being present in as the adaptability of affinity ligand The species and isotype in any immunoglobulin Fc domain in body.Albumin A can be used for purifying to be based on people γ 1, γ 2 or the heavy chains of γ 4 Antibody (for example, with reference to Lindmark et al., 1983J.Immunol.Meth.62:1-13).Protein G is recommended to be used for all small (for example, with reference to Guss et al., 1986EMBO is J.5 by mouse isotype and people γ 3:1567-1575).Base attached by affinity ligand Matter is most frequently agarose, but other matrix also can be used.Matrix (such as controlled pore glass or the poly- (benzene of mechanical stability Ethene divinyl) benzene) to may achieve compared with using the faster flow velocity of agarose and shorter processing time.If antibody includes C_H3Domain, then Bakerbond ABX^TMResin (J.T.Baker, Phillipsburg, N.J.) can be used for purifying.Depending on being intended to back The antibody of receipts, other Protein purification techniques, such as ion exchange column classification separation, ethanol precipitation, reversed-phase HPLC, silicon also can be used Glue chromatogram, heparin SEPHAROSE^TMChromatogram, anion or cationic ion-exchange resin (such as poly-aspartate post) chromatogram, chromatogram are gathered Burnt, SDS-PAGE and ammonium sulfate precipitation.

After any any preliminary purification step, elution buffers of the pH between about 2.5 to 4.5 can be used to including target The mixture of antibody and pollutant implements low pH hydrophobic interaction chromatographs, and it is generally in low salt concn (for example, about 0-0.25M Salt) under implement.

Also be included in as defined herein it is low, in and high stringent conditions under with by coding the present invention antibody or antibody piece All or part of (such as part of the encoding variable regions) hair for the nucleotide sequence that the polynucleotide sequence of the separation of section represents The nucleic acid of raw hybridization.Generally long at least 15 (for example, 20,25, the 30 or 50) nucleosides of the hybridization portion of hybrid nucleic acid Acid.The hybridization portion of hybrid nucleic acid and coding anti-il-23 P19 polypeptides (for example, heavy chain or light chain variable district) or the core of its complement The some or all of sequence at least 80% (for example, at least 90%, at least 95% or at least 98%) of acid is consistent.It is described herein The hybrid nucleic acid of type can be used as (such as) cloning probe, primer (such as PCR primer) or diagnostic probe.

Some embodiments include the polynucleotide of separation, and it is SEQ ID NO comprising encoding amino acid sequence:84、86、 88th, 90,91,93,95,97,99,101,103,105,107,109,111,113,115,117 or 119 antibody of any one or The sequence of antibody fragment；And with polynucleotide sequence SEQ ID NO:83、85、87、89、92、94、96、98、100、102、104、 106th, 108,110,112,114,116 or 118 at least 80%, at least 90%, at least 95%, at least 98% or at least 99% are consistent Sequence.

Some embodiments include the polynucleotide of separation, and it is SEQ ID NO comprising encoding amino acid sequence:158、 160th, 162 or 164 antibody of any one or the sequence of antibody fragment；And with polynucleotide sequence SEQ ID NO:157、159、 The consistent sequence of 161 or 163 at least 80%, at least 90%, at least 95%, at least 98% or at least 99%.

Some embodiments include the polynucleotide of separation, and it is SEQ ID NO comprising encoding amino acid sequence:121、 123rd, 125,127,128,130,132,134,136,138,140,142,144,146,148,150,152,154 or 156 times The antibody of one or the sequence of antibody fragment；And with polynucleotide sequence SEQ ID NO:120、122、124、126、129、131、 133rd, 135,137,139,141,143,145,147,149,151,153 or 155 at least 80%, at least 90%, at least 95%, extremely Few 98% or at least 99% consistent sequence.

Some embodiments include the polynucleotide of separation, and it is SEQ ID NO comprising encoding amino acid sequence:166、 168th, 170 or 172 antibody of any one or the sequence of antibody fragment；And with polynucleotide sequence SEQ ID NO:165、167、 The consistent sequence of 169 or 171 at least 80%, at least 90%, at least 95%, at least 98% or at least 99%.

Some embodiments include the polynucleotide of separation, and it includes coding light chain variable region amino acid sequence and amino acid Sequence SEQ ID NO:84、86、88、90、91、93、95、97、99、101、103、105、107、109、111、113、115、117 Or 119 any one at least 80%, at least 90%, at least 95%, at least 98% or at least 99% consistent antibody or antibody piece The sequence of section.Some embodiments include the polynucleotide of separation, and it includes coding light chain variable region amino acid sequence and amino Acid sequence SEQ ID NO:158th, any one of 160,162 or 164 at least 80%, at least 90%, at least 95%, at least 98% or The sequence of at least 99% consistent antibody or antibody fragment.Some embodiments include the polynucleotide of separation, and it includes coding Heavy chain variable amino acid sequence and amino acid sequence SEQ ID NO:121、123、125、127、128、130、132、134、 136th, any one of 138,140,142,144,146,148,150,152,154 or 156 at least 80%, at least 90%, at least 95%th, the sequence of at least 98% or at least 99% consistent antibody or antibody fragment.Some embodiments include the poly-nuclear of separation Thuja acid, it includes encoding heavy chain variable region amino acid sequence and amino acid sequence SEQ ID NO:166th, 168,170 or 172 times The sequence of one at least 80%, at least 90%, at least 95%, at least 98% or at least 99% consistent antibody or antibody fragment.

Terms used herein " consistent " or " uniformity percentage " two or more nucleic acid or peptide sequence up and down Refer in text two or more sequences or subsequence carry out to when align (to obtain maximum correspondence) when it is identical or have The nucleotides or amino acid residue of prescribed percentage are identical.It is to determine uniformity percentage, sequence alignment is optimal right to reach Than purpose (for example, room can be introduced in the first amino acid or nucleotide sequence to reach most with the second amino acid or nucleotide sequence Good alignment).Then the amino acid residue or nucleotides at orresponding amino acid position or nucleotide position are contrasted.If in First ray Position occupied with the correspondence position in the second sequence by same amino acid residue or nucleotides, then the molecule is in the opening position Unanimously.Uniformity positional number purpose function of the uniformity percentage common to the sequence between two sequences is (i.e., unanimously Property %=uniformity positional number/total positional number (for example, lap position number) x100).In some embodiments, two are contrasted Sequence (for example, not including extending the additional sequences outside contrast sequence) length phase behind introducing room in sequence when suitable Together.For example, when contrasting variable region sequences, targeting sequencing and/or constant domain sequence are not considered.Sequence between two sequences In contrast, " correspondence " CDR refers to the CDR (for example, CDR-H1 of each sequence) in two sequences at same position.

Mathematical algorithm can be used to complete in the measure of uniformity percentage or Similarity Percent between two sequences.For Contrast two sequences mathematical algorithm preferred non-limiting examples be Karlin and Altschul algorithm (1990, Proc.Natl.Acad.Sci.USA 87:2264-2268), it is according to Karlin and Altschul, and 1993, Proc.Natl.Acad.Sci.USA 90:5873-5877 is changed.This algorithm has included Altschul's et al. (1990, J.Mol.Biol.215 in NBLAST and XBLAST programs:403-410).BLAST nucleotide searches can use NBLAST journeys Sequence (fraction=100, word length=12) is implemented, so as to obtain the nucleotide sequence homologous with the nucleic acid of encoding target albumen. BLAST protein searches can use XBLAST programs (fraction=50, word length=3) to implement, homologous with target protein so as to obtain Amino acid sequence.For comparative purposes, room alignment is added to obtain, can be such as Altschul et al., 1997, Nucleic Acids Res.25:Gapped BLAST are utilized described in 3389-3402.Or PSI-Blast can be used to implement iterative search, Teleconnexion (same document) between its detection molecules.Utilizing BLAST, Gapped BLAST and PSI-Blast programs When, each program (for example, XBLAST and NBLAST) parameter preset can be used.For contrasting the another preferably non-limiting of sequence Example is Myers and Miller algorithm (CABIOS (1989)).This algorithm has been included in ALIGN programs (2.0 editions), and it is A part for GCG sequence alignment software kits.When using alignment program to contrast amino acid sequence, it can be used PAM120 weightings residual Poor table, the GAP LENGTH PENALTY equal to 12 and the gap penalty equal to 4.Other algorithms for sequence analysis are known in the art And including ADVANCE and ADAM, such as Torellis and Robotti, 1994, Comput.Appl.Biosci.10:Described in 3-5； And FASTA, such as Pearson and Lipman, 1988, Proc.Natl.Acad.Sci.USA 85:Described in 2444-8.In FASTA Interior, ktup is the control option of setting sensitivity and search speed.If ktup=2, by checking alignment residue to finding Contrast the similar area in two sequences；If ktup=1, the alignment amino acid of checklist one.For protein sequence, can incite somebody to action Ktup is set as 2 or 1, or is set as 1 to 6 for DNA sequence dna.If not specified ktup, for albumen default value be 2 and for DNA default values are 6.Or CLUSTAL W algorithms can be used to implement for protein sequence alignment, such as Higgins et al., 1996, Methods Enzymol.266:Described in 383-402.

Non-therapeutic use

Antibody described herein can be used as affinity purification agent.In this method, using it is well known that method fixes antibody Such as Protein A resin in solid phase.Sessile antibody is set to be connect with the sample containing IL-23P19 albumen to be purified (or its fragment) Touch, and then use suitable solvent wash vehicle, thus removed in removable sample the IL-23P19 albumen that is combined with sessile antibody with Outer essentially all material.Finally, with another suitable solvent wash vehicle, so as to discharge IL-23P19 albumen from antibody.

Anti-il-23 p 19 antibodies, for example, humanization anti-il-23 p 19 antibodies also suitable for diagnostic analysis with detection and/or Quantitative determine the IL-23 expression in IL-23 albumen, such as detection specific cells, tissue or serum.Anti-il-23 p 19 antibodies can examine Disconnected property be used for such as monitoring of diseases show or be in progress as a part for clinical trial program with for example determine to give treat and/ Or the effect of prevention scheme.Detection can be promoted by being coupled anti-il-23 p 19 antibodies.The example of detectable substance includes each Kind of enzyme, prothetic group, fluorescent material, luminescent substance, bioluminescence material, radioactive substance, taken the photograph using various positron emission faults The positron emitting metal and on-radiation paramagnetic metal ion of shadow art.On can be with the diagnosticum of being suitable for of the present invention The metal ion of antibody binding, see, for example, U.S. Patent No. 4,741,900.

Anti-il-23 p 19 antibodies can be used for diagnosis IL-23 associated conditions (such as to be characterised by the disease of IL-23 abnormal expression Disease) or determine in the whether increased method of risk that IL-23 associated conditions occurs in individual.These methods include making from individual Biological sample contacts with IL-23p19 antibody and detects antibody and IL-23p19 combination." biological sample " refers to from individual, carefully Any biological sample that other cell deriveds of born of the same parents system, tissue culture or potential expression IL-23 obtain.For from mammal The method for obtaining tissue biopsy and body fluid is well known in the art.

In some embodiments, this method can comprise additionally in IL-23 contents and the control sample compared in Patient Sample A IL-23 contents in (such as not suffering from the individual of IL-23 associated conditions), to determine whether patient suffers from IL-23 associated conditions Or in the risk for showing IL-23 associated conditions.

In some embodiments, such as diagnostic purpose, it may be advantageous to detectable part labelled antibody.It can make With a variety of detectable labels, including radio isotope, fluorescence labeling, zymolyte mark etc..A variety of known technologies can be used to make Mark is conjugated with antibody indirect.For example, antibody and biotin-conjugated can be made, and any in three major types mark described above can be made Kind be conjugated with avidin, or vice versa it is as the same.Biotin is optionally combined with avidin, and the mark is thus Can be with the indirect mode and antibody conjugate.Or to reach the indirect conjugation of mark and antibody, antibody can be made with resisting compared with smaller part Former (such as digoxin (digoxin)) is conjugated and makes a kind of different types of above-mentioned mark with antihapten antibody (for example, anti-ly Digoxin antibody) it is conjugated.Thus, it may achieve the indirect conjugation of mark and antibody.

Exemplary radioisotope mark includes³⁵S、¹⁴C、¹²⁵I、³H and¹³¹I.Use (such as) described in documents below Technology can use labelled with radioisotope antibody：Current Protocols in Immunology, volume 1 and 2,1991, Coligen et al. is edited, Wiley-Interscience, New York, N.Y., Pubs.Can by (such as) scinticounting come Measure radioactivity.

Exemplary fluorescence mark includes deriving from following mark：Rare Earth Chelate (Europium chelate) or fluorescein and its derivative Thing, rhodamine (rhodamine) and its derivative, red sulphonyl (dansyl), Liz amine (Lissamine), phycoerythrin and Ke Sasi red (Texas Red).Fluorescence labeling and antibody conjugate, such as Current Protocols can be made via known technology Those disclosed in Immunology (above-mentioned document).Fluorescence photometer can be used to carry out quantitative measurment fluorescence.

It is known in the art it is a variety of through fully characterizing enzyme-substrate mark (for example, summary is referring to U.S. Patent No. 4,275, No. 149).Enzyme is typically catalyzed the chemical modification that various technology measurements can be used in chromophoric substrate.For example, change can be can be with The substrate colors change of spectrophotometer method measurement.Or enzyme can change fluorescence or the chemiluminescence of substrate.Quantify change in fluorescence Technology as described above.Chemical luminous substrate can by chemical reaction through electron excitation, can then launch it is usable (such as) The light of chemiluminescence meter measurement provides energy to fluorescent receptor.

The example of enzyme mark includes luciferase (such as Fluc and bacteriofluorescein enzyme, U.S. Patent No. No. 4,737,456), luciferin, 2,3- dihydros phthalazine diketone, malic dehydrogenase, urase, peroxidase (such as horseradish mistake Oxide enzyme (HRPO)), alkaline phosphatase, beta galactosidase, glucoamylase, lysozyme, carbohydrate oxidase (such as glucose Oxidizing ferment, galactose oxidase and glucose-6-phosphate dehydrogenase (G6PD)), Heterocyclic oxidases (such as uricase and xanthine oxidation Enzyme), lactoperoxidase, microperoxisome etc..The technology of conjugated enzyme and antibody be set forth in (such as) in documents below：O' Sullivan et al., 1981, Methods for the Preparation of Enzyme-Antibody Conjugates (J.Langone and H.Van Vunakis are compiled for use in Enzyme Immunoassay, Methods in Enzym. Volume), Academic press, N.Y., 73：147-166.

Enzyme-substrate combination example include (such as)：Horseradish peroxidase (HRPO) and the hydrogen peroxide as substrate Enzyme, wherein catalase oxidation dye precursors (such as o-phenylenediamine (OPD) or 3,3', 5,5'- tetramethyl biphenyl amine hydrochlorates (TMB))；Alkaline phosphatase (AP) and the p-nitrophenyl phosphate as chromophoric substrate；And beta-D-galactosidase (β-D-Gal) With chromophoric substrate (such as p-nitrophenyl-beta-D-galactosidase or fluorogenic substrate 4-methyl umbelliferone-β-D- galactosides Enzyme).

Those skilled in the art can be appreciated that various other enzyme-substrate combinations.The general summary of the combination is special referring to the U.S. Sharp No. 4,275,149 and U.S. Patent No. 4,318,980.

In another embodiment, using unmarked humanization anti-il-23 P19 antibody and with combining the anti-IL- of the humanization The labeled antibody of 23P19 antibody detects.

Antibody described herein can be used for any known analysis methods in, such as competitive binding analysis, directly and indirectly folder The heart (sandwich) is analyzed and immunoprecipitation analysis.For example, with reference to Zola, Monoclonal Antibodies：A Manual Of Techniques, the 147-158 pages (CRC Press companies, 1987).

Anti-il-23 p 19 antibodies or its antigen-binding fragment can be used for the combination for suppressing IL-23 and DCRS5.These sides Method includes giving anti-il-23 p 19 antibodies or its antigen-binding fragment to cell (such as mammalian cell) or cellular environment, by This suppresses the signal transduction mediated by DCRS5.These methods in vitro or can be carried out in vivo." cellular environment " is Refer to tissue, culture medium or the extracellular matrix for surrounding cell.Cytotropic cellular environment gives anti-il-23 p 19 antibodies or it is anti- Former binding fragment, to enable the antibody or fragment with being combined in the IL-23 molecules of outside and encircling cell, so as to hinder Only IL-23 is combined with its acceptor.

Diagnostic kit

Anti-il-23 P19 antibody can be used in diagnostic kit, i.e. scheduled volume reagent and the specification of implementation diagnostic analysis Packaged combination.If with enzymic-labelled antibody, then kit may include that substrate needed for enzyme and co-factor (such as provide detectable hair Color group or the substrate precursor of fluorogen).In addition, it may include other additives, such as stabilizer, buffer (such as blockade buffering Agent or lysis buffer) etc..The relative quantity of various reagents can significantly change so that the concentration of the reagent in the solution can be notable Optimize the sensitivity of analysis.Reagent (generally freeze-dried) can provide in dry powder form, and it includes that reagent solution can be made upon dissolution Excipient with suitable concentration.

Therapeutic use

In another embodiment, humanization anti-il-23 P19 antibody disclosed herein can be used for treatment as described herein each The kind illness relevant with IL-23P19 expression.For treat IL-23 associated conditions method include to individual in need to Give the humanization anti-il-23 p 19 antibodies of therapeutically effective amount.

Humanization anti-il-23 P19 antibody or medicine are given by any suitable way, including parenteral, subcutaneous, peritonaeum Interior, intrapulmonary and intranasal, and if desired local immunosuppression is treated, then implement to give in focus (including perfusion or otherwise Graft is set to be contacted before transplantation with antibody).Humanization anti-il-23 P19 antibody or medicine can (such as) infusion or inject shape Formula is given.Parenteral infusions include intramuscular, intravenous, intra-arterial, intraperitoneal or subcutaneous administration.In addition, the anti-IL- of humanization 23P19 antibody is suitably given by pulse infusion, is especially given with the attenuated dosage of antibody.In one aspect, note is passed through Penetrate to implement to be administered, be most preferably administered by intravenous or subcutaneous injection, be to give in short term also this depend partly on giving It is to give for a long time.

When preventing or treating disease, the optimal dose of antibody may depend on many factors, such as be intended to as hereinbefore defined Treating type, the order of severity of disease and the course of disease, the purpose for giving antibody of disease is preventative or therapeutic, previously controlled Treatment, patient clinical history and the reaction to antibody, and the decision of attending doctor.Antibody disposably give or through it is a series for the treatment of with Appropriate ways give patient.

Depending on disease type and the order of severity, no matter (such as) by separately giving one or more times or by continuous defeated Note to give, be about 1 μ g/kg to 20mg/kg (such as 0.1-15mg/kg) to the antibody initial candidate dosage that patient gives.Take Certainly can be between about 1 μ g/kg to 100mg/kg or more in high scope in factor referred to above, typical daily dose.For through number It or longer time repeat to give, and depending on the patient's condition, continued treatment is until desired suppression occur in disease symptomses.However, can Use other dosages.The progress of this therapy can easily be monitored by common technology and analysis.Exemplary dose scheme is public Open in WO 94/04188.

Terms used herein " suppression " in " improvement " and " mitigation " identical background with meaning to reduce the one or more of disease Individual feature.

Mode that can be consistent with good medical practice is allocated to antibody compositions, dosage and is given.Herein up and down Wen Zhongxu consider factor include treated particular condition, the specific mammal treated, individual patient the clinical patient's condition, The cause of disease, the delivering site of medicine, administration way, give arrangement of time and the other factors known to practitioners.Antibody to be given " therapeutically effective amount " may depend on the factor, and for prevention, improve or illness that treatment is relevant with IL-23 expression needed for Minimum.

Antibody need not (but optionally) allocated together with the medicine that one or more are currently used in prevention or treat the illness.Institute The effective dose for stating other drugs depends on the class of amount, illness or treatment that humanization anti-il-23 P19 antibody is present in composite Type and above-mentioned other factors.The other drugs are generally with identical dosage used above and giving approach to use or to be upper About 1% to 99% of dosage used in text.

IL-23 associated conditions

Anti-il-23 p 19 antibodies or medicament are characterised by that for example (such as lymph is thin because of immunocyte suitable for treatment or prevention Born of the same parents or BMDC) activation it is improper caused by IL-23 abnormal expressions immune disorders.This IL-23 unconventionality expression is attributable to Such as IL-23 protein contents increase.Anti-il-23 p 19 antibodies or its antigen-binding fragment also can be used for treating or preventing breathing system System illness, dysbolism (such as diabetes) and some cancers.Immune disorders are treated or prevented according to method described herein, exhaled Desorption system illness, dysbolism or cancer, it is by needing this individual treated or prevented to give the anti-IL- of effective dose 23p19 antibody or medicament, thus antibody the IL-23 related to morbid state activity is reduced to reach.

It is characterized as the improper activation of immunocyte and can come the immunological diseases treated or prevented by methods described herein According to (such as) type of the basic hypersensitivity of the illness classifies.The reaction is commonly divided into four classes：Allergic reaction, Cytotoxicity (cytolytic) reaction, immune complex reaction or cell-mediated immune (CMI) reaction (are also referred to as delayed Super quick (DTH) reacts).(for example, with reference to Fundamental Immunology, (William E.Paul are edited, Raven Press, N.Y., the 3rd edition, 1993)).Immunological diseases include inflammatory disease and autoimmune disease.

The instantiation of the immunological diseases includes following disease：Rheumatoid arthritis, LADA demyelinate disease Sick (for example, multiple sclerosis, allergic encephalomyelitis), endocrine ophthalmopathy, uveoretinitis, generalized erythema wolf Sore, myasthenia gravis, Graves' disease (Grave's disease), glomerulonephritis, autoimmune hematological disease, inflammatory Enteropathy (for example, Crohn's disease or ulcerative colitis), allergic reaction, allergic reaction, Sjogren Cotards, I types sugar Urinate disease, PBC, Wei Genashi granulomatosis (Wegener's granulomatosis), fibromyalgia, Polymyositis, dermatomyositis, inflammation myositis, Multiple Endocrine deficiency, Schmidt's syndrome (Schmidt's Syndrome), Autoimmune uveitis, Addison's disease, paranephritis, thyroiditis, Hashimoto's thyroiditis, itself Autoimmune thyroid disease, pernicious anaemia, lipogastry, chronic hepatitis, lupoides hepatitis, atherosclerosis, Subacute Cutaneous Lupus erythematosus, hypoparathyroidism, Dresslar Cotard (Dressler's syndrome), LADA blood Platelet reduces disease, ITP, hemolytic anemia, pemphigus vulgaris, pemphigus, dermatitis herpetiformis, spot Bald, pemphigoid, scleroderma, progressive systemic sclerosis disease, CREST syndromes (calcinosis, Raynaud's phenomenon (Raynaud's phenomenon), oesophagus activity are bad, sclerodactyly and capillarectasia), male and female autoimmunity Property infertility, ankylosing spondylitis, ulcerative colitis, MCTD, PAN, systemic gangrenosum acne Vasculitis, atopic dermatitis, atopic rhinitis, Goodpasture Cotard (Goodpasture's syndrome), just plus Si Shi diseases (Chagas'disease), sarcoidosis, rheumatic fever, asthma, recurrent miscarriage, antiphospholipid syndrome, farmer lung (farmer's lung), erythema multiforme, postcardiotomy syndrome, Cushing's syndrome (Cushing's syndrome), from Body autoimmune chronic active hepatitis, bird-breeder's lung, toxic epidermal necrolysis disease, Alport's syndrome (Alport' S syndrome), pulmonary alveolitis, sequoiosis, FA, interstitial lung disease, erythema nodosum, gangraenosum purulence skin Disease, transfusion reaction, takayasu's arteritis (Takayasu's arteritis), polymyalgia rheumatica, temporal arteritis, snail fever, Giant cell arteritis, roundworm disease, aspergillosis, aspirin triad (Sampter's syndrome), eczema, lymthoma sample Granulomatosis, Behcet's disease (Behcet's disease), caplan syndrome (Caplan's syndrome), Kawasaki disease (Kawasaki's disease), dengue fever (dengue), encephalomyelitis, endocarditis, davie's disease, intraocular Inflammation, erythema elevatum diutinum, psoriasis, psoriatic arthritis, fetal erythrocytosis, eosinophilic fasciitis, Post-transfusion purpura syndrome (Shulman's syndrome), Felty's syndrome (Felty's syndrome), filariasis, eyelash Shape body inflammation, chronic cyclitis, heterochromatic cyclitis, Fox's cyclitis (Fuch's cyclitis), IgA nephrosis, henry- It is comprehensive to give up Er Shi purpuras (Henoch-Schonlein purpura), graft versus host disease(GVH disease), graft rejection, cardiomyopathy, myasthenia Simulator sickness (Eaton-Lambert syndrome), relapsing polychondritis, cryoglobulinemia, Walden Si Telun macroglobulin Mass formed by blood stasis (Waldenstrom's macroglobulemia), Evan's syndome (Evan's syndrome), acute respiration are embarrassed Compel syndrome, lung inflammation, osteoporosis, delayed allergy and LADA gonadal failure.

In some embodiments, immune disorders are the immune disorders of T cell mediation, and therefore anti-as described herein IL-23p19 antibody and medicament are also applied to treat or prevent the immune disorders of T cell mediation.

In an aspect, anti-il-23 p 19 antibodies or medicament are applied to treat or prevent the breathing of IL-23 unconventionality expressions System condition.Respiratory disorder is treated or prevented according to method described herein, is by the way that this is treated or prevented to needs Individual gives the anti-il-23 p 19 antibodies or medicament of effective dose, and thus antibody reduces the IL-23 activity related to morbid state Come what is reached.These illnesss include but is not limited to：Diseases of respiratory system, the obstructive lung disease of various causes, the lung of various causes Wind-puff, restrictive lung disease, interstitial lung disease (interstitial pulmonary disease/interstitial lung Disease), (adult respiratory distress syndrome integrates by cystic fibrosis, the bronchitis of various causes, bronchiectasis, ARDS Sign) and form of ownership pulmonary edema；Obstructive lung disease, asthma selected from COPD (chronic obstructive pulmonary disease), bronchial astehma, youngster Section's asthma, severe asthma, acute asthmatic attack and chronic bronchitis；Originating from COPD (chronic obstructive pulmonary disease) or α 1- eggs The pulmonary emphysema that white enzyme inhibitor lacks；Restrictive lung disease selected from sequoiosis, the harmful substance initiation by work correlation Restrictive lung disease (such as asbestosis (asbestosis) or anthraco-silicosis (silicosis)) and by lung neoplasm (such as carcinous leaching Hand shaft disease (lymphangiosis carcinomatosa), bronchovesicular cancer and lymthoma) caused by limit；By infecting, example The pneumonia as caused by virus, bacterium, fungi, protozoan, worm or other pathogenic infections；By various factors, such as air-breathing And pneumonia caused by left heart insufficiency；The pneumonia or fibrosis of radiation-actuate, collagen disease are (such as lupus erythematosus, systemic hard Skin disease or sarcoidosis), granuloma (such as Boeck's disease (Boeck's disease), idiopathic interstitial pneumonia or idiopathic Pulmonary fibrosis (IPF))；Mucus obstruction disease (mucoviscidosis), bronchitis, allergy as caused by bacterium or virus infection Property bronchitis and Poisoning bronchitis；Bronchiectasis；Pulmonary edema, for example, suction noxious material and foreign substance it Toxic pulmonary edema afterwards；Rhinitis, arthritis and associated joint disease, psoriasis, myelomatosis, multiple sclerosis, A Zi Extra large Mo's disease (Alzheimer's disease), glomerulonephritis and chronic atopic dermatitis.

In another aspect, anti-il-23 p 19 antibodies and medicament as described herein are also applied to treatment IL-23 exception tables The cancer reached.

Can be included by the IL-23 expressivities cancer of method described herein treatment (such as) leukaemia, such as it is acute white Blood disease, acute lymphatic leukemia, acute myelocytic leukemia are (for example, myeloblastosis, promyelocyte Leukaemia, myelomonocytic leukemias, monocytic leukemia or erythroleukemia), chronic leukemia, chronic myelocytic Property (granulocytic) leukaemia or chronic lymphocytic leukemia；Polycythemia vera；Lymthoma is (for example, Huo Qijin Family name's disease or Non-Hodgkin lymphoma)；Huppert's disease, Walden Si Telunshi macroglobulinemias；Heavy chain disease；Entity tumor, Such as sarcoma and carcinoma are (for example, fibrosarcoma, myxosarcoma, embryonal-cell lipoma, chondrosarcoma, osteogenic sarcoma, osteosarcoma, ridge Rope knurl, angiosarcoma, endotheliosarcoma, lymphangioendothelial sarcoma, lymphangioendothelial sarcoma, synovialoma, celiothelioma, outstanding Yin Shi tumours (Ewing's tumor), leiomyosarcoma, rhabdomyosarcoma, colon cancer, colorectal cancer, cancer of pancreas, breast cancer, oophoroma, Prostate cancer, squamous cell carcinoma, basal-cell carcinoma, gland cancer, syringocarcinoma, carcinoma of sebaceous glands, papillary carcinoma, papillary adenocarcinoma, capsule gland Cancer, cephaloma, bronchiolar carcinoma, clear-cell carcinoma, liver cancer, cholangiocarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Willms Family name's tumour (Wilms'tumor), cervical carcinoma, uterine cancer, testicular tumor, lung cancer, ED-SCLC, non-small cell lung cancer, carcinoma of urinary bladder, Epithelioma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, blood vessel are female Cytoma, acoustic neurinoma, oligodendroglioma, meningioma (menangioma), melanoma, neuroblastoma, view Film blastoma, nasopharyngeal carcinoma or the cancer of the esophagus).

Pharmaceutical composition and its administration

Composition comprising IL-23p19 bonding agents (such as anti-il-23 p 19 antibodies) can be given with immune disorders, exhale Desorption system illness or cancer or the individual in the risk with immune disorders, respiratory disorder or cancer.It is of the invention another Outer offer IL-23P19 bonding agents (for example, anti-il-23 P19 antibody) are preparing the cancer for preventing or treating expression IL-23P19 Purposes in the medicine of disease or immune disorders.Terms used herein " individual " means that any food in one's mouth of IL-23P19 bonding agents can be given Newborn animal patient, including (such as) people and non-human mammal, such as primate, rodent and dog.Clearly it is intended to The individual treated using methods described herein includes people.In the prevention or treatment of immune disorders or the cancer for expressing IL-23P19 In, antibody or medicine can be given individually or with other component combines.These components that can be given with antibody or pharmaceutical agent combinations include MTX (MTX) and immunomodulator, such as antibody or small molecule.

It comprising chain variable region amino acid sequence is SEQ ID that example for the antibody in these pharmaceutical compositions, which is, NO:84th, 86,88,90,91,93,95,97,99,101,103,105,107,109,111,113,115,117 or 119 any The humanized antibody of person or the antibody of antibody fragment.Example for the antibody in these pharmaceutical compositions is also can comprising heavy chain Change region amino acid sequence is SEQ ID NO:121、123、125、127、128、130、132、134、136、138、140、142、 144th, 146,148,150,152,154 or 156 humanized antibody of any one or the antibody of antibody fragment.

It comprising chain variable region amino acid sequence is SEQ that other examples for the antibody in these pharmaceutical compositions, which are also, ID NO:158th, 160,162 or 164 humanized antibody of any one or the antibody of antibody fragment.For these pharmaceutical compositions In preferred antibody be also comprising heavy chain variable amino acid sequence be SEQ ID NO:166th, any one of 168,170 or 172 Humanized antibody or antibody fragment antibody.

Other examples for the antibody in these pharmaceutical compositions are also to be comprising light chain variable district and weight chain variable district SEQ ID NO:160 and 166, SEQ ID NO:160 and 168, SEQ ID NO:158 and 166 or SEQ ID NO:158 and 168 The humanized antibody of any one or antibody fragment antibody.

It comprising light chain region amino acid sequence is SEQ ID that other examples for the antibody in these pharmaceutical compositions, which are also, NO:The antibody of 174 or 180 humanized antibody of any one.For the preferred antibody in these pharmaceutical compositions also be comprising Heavy chain variable amino acid sequence is SEQ ID NO:The antibody of 176 or 178 humanized antibody of any one.

Other examples for the antibody in these pharmaceutical compositions are also to include antibody A, antibody B, antibody C or antibody D Antibody.

It is known and a variety of delivery systems can be used to give IL-23P19 bonding agents.Introducing method is including but not limited to true Intracutaneous, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, Epidural cavity and peroral route.IL-23P19 bonding agents can by (such as) It is transfused, injects or injects to give, and can be given together with the other biological activity agent such as chemotherapeutic agent.Giving to be It is systemic or administer locally to.In preferred embodiments, given by being subcutaneously injected.Can (such as) in pre-filled syringe The composite for the injection is prepared, it can give once week about.

In a particular embodiment, IL-23P19 binding agent compositions are by injection, by conduit, by suppository or borrow Implant is helped to give, for the implant to have hole, non-porous or gel-like material, it includes the film such as silicone rubber membrane or fiber. Generally, when giving composition, anti-il-23 P19 antibody or the not adsorbable material of medicine are used.

In other embodiments, anti-il-23 P19 antibody or medicine are delivered with controlled release system.In one embodiment, Can be used pump (for example, with reference to Langer, 1990, Science 249:1527-1533；Sefton, 1989, CRC Crit.Ref.Biomed.Eng.14:201；Buchwald et al., 1980, Surgery88:507；Saudek et al., 1989, N.Engl.J.Med.321:574).In another embodiment, polymeric material can be used.(for example, with reference to Medical Applications of Controlled Release (Langer and Wise are edited, CRC Press, Boca Raton, Fla., 1974)；Controlled Drug Bioavailability, Drug Product Design and Performance (Smolen and Ball are edited, Wiley, New York, 1984)；Ranger and Peppas, 1983, Macromol.Sci.Rev.Macromol.Chem.23:61.Also referring to Levy et al., 1985, Science 228:190； During et al., 1989, Ann.Neurol.25:351；Howard et al., 1989, J.Neurosurg.71:105.) other controls Release system be discussed in (such as) in Langer (above-mentioned document).

IL-23P19 bonding agents (for example, anti-il-23 P19 antibody) can include the bonding agent of therapeutically effective amount and one or more The pharmaceutical compositions of the composition of kind of pharmaceutically compatible are given.

In typical embodiments, pharmaceutical composition is allocated as being applied to intravenously or subcutaneously give according to routine operation The pharmaceutical composition of people.Generally, it is the solution that is present in the water-based buffer solution of sterile isotonic that injection, which is given with composition,.If need Will, medicine also may include solubilizer and local anesthetic (such as lignocaine (lignocaine)) to mitigate the pain of injection site Bitterly.Generally, each composition individually or is mixed and supplied with unit dosage forms, for example, as lyophilized dry powder or without the form of aqueous concentrate It is present in the sealing container of the amount of lined out activity agent such as ampoule or anther sac.If medicine is gives by being transfused, then It can be distributed into the infusion bottle containing sterile pharmaceutical grade water or salt solution.If medicine is given by injecting, then can provide Ampoule containing Injectable sterile water or salt solution is make it that each composition can be mixed before giving.

Furthermore, it is possible to provide pharmaceutical composition in the form of pharmaceutical kit, the kit includes (a) and contained in lyophilized The container and (b) of the IL-23P19 bonding agents (for example, anti-il-23 P19 antibody) of form contain pharmaceutically acceptable injection The second container of diluent (for example, sterilized water).Pharmaceutically acceptable diluent can be used for restructuring or dilution to freeze anti-IL- 23P19 antibody or medicine.Optionally, the container can be accompanied with the political affairs of preparation, use or the sale of supervision medicine or biological product The bulletin of mansion mechanism defined form, the bulletin show that government organs' approved is used for preparation, use or sale that people gives.

IL-23P19 bonding agents (for example, anti-il-23 P19 antibody) are treating or preventing immune disorders or expression IL-23P19 Cancer in effective dose can be determined by standard clinical techniques.In addition, optionally it can be assisted in using ex vivo assay Optimal dose scope.Precise dosage used may also depend on the stage of the approach of giving and immune disorders or cancer in allotment, and should Determined according to the situation of the judgement of practitioners and each patient.Can according to derived from vitro or animal model test system agent Quantitative response curve infers effective dose.

Generally, IL-23P19 cancer is given with immune disorders or expressed to anti-il-23 P19 antibody or IL-23P19 bonding agents The dosage of the patient of disease is typically about 0.1mg/kg to about 100mg/kg whose body weights.The dosage for giving individual is about 0.1mg/kg To about 50mg/kg whose body weights, about 1mg/kg to about 30mg/kg, about 1mg/kg to about 20mg/kg, about 1mg/kg to about 15mg/ Kg or about 1mg/kg are to about 10mg/kg.

Exemplary dose includes but is not limited to 1ng/kg to 100mg/kg.In some embodiments, dosage is about 0.5mg/kg, about 1mg/kg, about 2mg/kg, about 3mg/kg, about 4mg/kg, about 5mg/kg, about 6mg/kg, about 7mg/kg, about 8mg/kg, about 9mg/kg, about 10mg/kg, about 11mg/kg, about 12mg/kg, about 13mg/kg, about 14mg/kg, about 15mg/kg or About 16mg/kg.The dosage can be for example following frequency give：Daily, (weekly), twice a week, often once in a week Zhou Sanci, it is secondary on every Thursdays, secondary on every Fridays, secondary on every Saturdays, once every two weeks or monthly, each two moon once or every three months Once.In a particular embodiment, dosage be about 0.5mg/kg/ weeks, about 1mg/kg/ weeks, about 2mg/kg/ weeks, about 3mg/kg/ Week, about 4mg/kg/ weeks, about 5mg/kg/ weeks, about 6mg/kg/ weeks, about 7mg/kg/ weeks, about 8mg/kg/ weeks, about 9mg/kg/ weeks, about 10mg/kg/ weeks, about 11mg/kg/ weeks, about 12mg/kg/ weeks, about 13mg/kg/ weeks, about 14mg/kg/ weeks, about 15mg/kg/ weeks or About 16mg/kg/ weeks.In some embodiments, dosage is in the scope of about 1mg/kg/ weeks to about 15mg/kg/ weeks.

In some embodiments, the pharmaceutical composition comprising IL-23P19 bonding agents can also include to be conjugated with the bonding agent Or unconjugated medicine.Anti-il-23 P19 antibody or IL-23P19 bonding agents can be exempted from one or more for treatment or prevention The combination of the medicine of epidemic disease disease or cancer is given jointly.

The combination treatment is given that disease parameters (for example, severity of symptoms, symptom number or recurrence frequency) can be produced and added up Or cooperative effect.

The therapeutic scheme given for combination, in one embodiment, by anti-il-23 P19 antibody or IL-23P19 Bonding agent is given simultaneously with medicine.In another embodiment, anti-il-23 P19 antibody or IL-23P19 are being given Medicine is given before or after bonding agent, i.e., before or after anti-il-23 P19 antibody or IL-23P19 bonding agents is given At least one hour and most long several months, for example, at least one hour, 5 hours, 12 hours, 1 day, 1 week, 1 month or 3 months are given and treated Medicine.

Product

In another aspect, including the product available for the material for treating above-mentioned illness is contained.The product include container and Mark.Appropriate vessel include (such as) bottle, bottle, syringe and test tube.Container can be from a variety of materials such as glass or plastics Material is formed.Container accommodates the composition that can effectively treat the patient's condition and can have sterile gateway.For example, container can be intravenous molten Liquid bag or the bottle that plug can be pierced into hypodermic needle.Active medicine in composition resists for humanization anti-il-23 P19 Body.The mark instruction being connected on container or with container, said composition are for treating the selected patient's condition.Product, which can also include second, to be held Device, the second container include pharmaceutically acceptable buffer solution, such as phosphate buffered saline (PBS), Ringer's solution (Ringer's ) and dextrose solution solution.It may also include considers desired other materials, including its according to business and user perspective His buffer solution, diluent, filter, pin, syringe and package insert with operation instruction.

The present invention is further described in the examples below that, and the embodiment is not intended to limit scope.

Embodiment

Embodiment 1：Produce humanization anti-il-23 p 19 antibodies

Mouse leader antibody 6B8 is changed into and is fitted together to by what 6B8 mouse variable domain and human constant IgG1KO domains formed Antibody.Mouse antibodies 6B8 is showed in table 1 above and table 2.IgG1KO (KO is knock out, is rejected) has two displacements prominent Become (Leu234Ala and Leu235Ala), the mutation by reducing effector function, such as Fc γ R and complement with reference to and eliminate ADCC and CDC activity.The variable domain of mouse antibodies is identical with the variable domain of chimeric antibody.Chimeric antibody is produced to confirm antibody Function and ensure to obtain correct sequence.Then the variable region humanization of antibody is made via design and screening technique.With any given The library of the mankind and mouse residues change can be prepared in position for the mode of the mankind or mouse residues.For human germline with it is small Those different amino acid, prepare such library between mouse antibody.Only selection retains the pure lines of the function of parent mouse antibody.It is anti- Body 6B8 representative humanization variable region is showed in table 5 and table 6.

In this way, antibody A, antibody B, antibody C and antibody D are to be grown by mouse antibodies 6B8 choosings to IgG 1-KO (KO =reject) humanized antibody obtained in/κ skeletons.Antibody A, B, C and D are showed in table 7.

Embodiment 2：The combination of antibody and the albumen of recombinant il-2 3

A mouse anti-il-23 p 19 antibodies and the recombinant human IL-23 dynamics combined and affinity (table 9)) are set out below. Single-column purifying after, using from fusion knurl caused by material, using Fortebio Octet (Fortebio, Menlo Park, CA dynamics and binding affinity are measured).Because Octet is not the technology based on fluidics, institute does not provide pair in this approach The accurate measure of dissociation rate.In some cases, the estimate of affinity can only be obtained.

Table 9

Antibody	ka(1/Ms)	kd(1/s)	KD(pM)
				18C4	3.84E+05	2.14E-06	5.57
18E 5	3.29E+05	2.61E-06	7.93
				18D3	3.19E+05	2.16E-06	6.78
20E8	4.21E+05	2.69E-04	638
				22E2	3.46E+05	3.53E-04	1024
24A5	2.02E+05	4.57E-06	22.6
				15C11	4.11E+05	1.07E-05	26
43F5	1.72E+05	5.96E-06	34.6
				27G8	1.57E+05	4.26E-06	27.2
31H9	2.99E+05	3.45E-06	11.5
				2D1		<1e-6	<1
9D12		<1e-6	<1
				6B8		<1e-6	<1
73H10	5.29E+04	5.24E-06	99.2
				74H3	3.06E+04	2.09E-06	68.3
35H8
				26F7	4.76E+05	1.34E-05	28.1
34G3	9.18E+05	3.10E-05	32.8
				34D9	3.44E+03	1.87E-06	544

B) measurement comes from the affinity of mouse antibodies 6B8 humanized antibody.Using ProteON XPR36 (Biorad, Hercules, CA) measurement and overall fit be into 1:The kinetic binding data of 1 binding model shows, with covalently couple p19 and The interaction of the recombinant il-2 3 (it contains the joint of 21 amino acid or does not contain the joint) of p40 subunits has high parent And power, the scope of affinity is between 1pM-100pM (table 10).Also test antibody 6H12 (is disclosed in WO 2007/027714 In), antibody QF20 (being disclosed in WO 2007/024846) and antibody C1273 (being disclosed in WO 2007/005955).

Table 10

C) with ProteON XPR36 measurement anti-il-23 p 19 antibodies and the stump-tailed macaque IL-23 affinity combined and dynamics Data, and overall fit is into 1:1 binding model (table 11).Also test antibody 6H12 (being disclosed in WO 2007/027714), anti- Body QF20 (being disclosed in WO 2007/024846) and antibody C1273 (being disclosed in WO 2007/005955).

Table 11

Antibody	K_D(pM)	k_a(1/Ms)	k_d(1/s)
				Antibody A	<1	2.95E+06	<1e-6
Antibody B	<1	2.99E+06	<1e-6
				Antibody C	2.9	3.23E+06	9.36E-06
Antibody D	15.9	2.07E+06	3.29E-05
				C-1273	>5,000	n/a	n/a
6H12	157	9.91E+05	1.56E-04
				QF20	1.2	3.90E+06	4.78E-06

D) relative to the molecule selectivity of human IL-12

Anti-il-23 p 19 antibodies are also injected in human IL-12 surface under 100nM concentration.Use Fortebio Octet The binding signal of these antibody measured is zero, and this shows these antibody selective binding mankind IL-23.Also in 50% mankind's blood The combination of anti-il-23 p 19 antibodies and IL-23 is analyzed in the presence of clear and observes that serum does not make significant difference to association rate, so as to Prove to combine and there is high specific.

Embodiment 3：The competitive binding assay that mankind IL-23 is combined with mankind IL-23R/Fc

Mankind IL-23R-Fc is caught on biosensor surface, and injects 10nM mankind IL-23.Sensor collection of illustrative plates refers to Show the specific binding (Fig. 2, top) between IL-23 and DCRS5.Antibody then with 10nM mankind's IL-23 co-injections The interaction that whether can suppress between IL-23 and DCRS5 is combined to assess antibody with IL-23.In this embodiment, if Antibody is combined with mankind IL-23 and can suppress to interact, then it will be observed that combining reduces or without with reference to (Fig. 2, bottom). In illustrated embodiment, antibody A and the 10nM recombinant human IL-23 co-injections of equimolar concentration.

Embodiment 4：Functional cell is analyzed, and suppress mouse boosting cell stimulates generation IL-17 through IL-23

A kind of functional cell for anti-il-23 p 19 antibodies is analyzed, and the monokaryon for measuring its suppression from mice spleen separation is thin The ability for the generation IL-17 that born of the same parents stimulate through IL-23.Human recombinant IL-23 albumen can stimulate IL-17 to be released from mouse boosting cell Put.In addition, the mankind IL-23 of the natural origin found in the supernatant of activation human monocyte's property THP-1 cells can be used for Mouse monokaryon cell is stimulated to produce IL-17.

Human recombinant IL-23 carrys out the natural mankind IL-23 of self-activation THP-1 cells and resisted with the anti-IL-23 p 19 through titration Body pre-incubation together.Then IL-23/ Antibody Combinations are added into the muroid splenocyte of fresh separated.Only recombinant il-2 3 is used Make positive control.Culture two days after, collect cell supernatant and by ELISA (R＆D Systems, Minneapolis, MN IL-17) is analyzed.The representative IC of anti-il-23 p 19 antibodies is set out below₅₀Value.Institute's test antibody is small as caused by fusion knurl Mouse antibody (1-19 arrange, referring to table 1 and table 2), chimeric antibody (20-23 row) and antibody A are to D.Also test antibody 6H12 (being disclosed in WO 2007/027714), antibody QF20 (being disclosed in WO 2007/024846) and antibody C1273 (are disclosed in WO In 2007/005955).

Table 12

Embodiment 5：Specific Function for IL-12 in the analysis of mankind's activating T cell is tested

Anti-il-23 p 19 antibodies are tested in the analysis of mankind's activating T cell to suppress IL-12 feature.Human recombinant IL-12 (1ng/ml) pre-incubation together with 5 μ g/ml anti-il-23 p 19 antibodies.Then the IL-12/ Antibody Combinations are added to In human T cells mother cell from PHA.RIL-12 is only used as positive control.Anti- IL-12p70 antibody (Bender MedSystems, Vienna, Austria) it is used as the inhibiting antibody of control.After culture two days, cell supernatant is collected And IFN-γ is analyzed by ELISA (R＆D Systems).Average value (the pg/ of triplicate test sample and measure IFN-γ ml).As a result it is showed in (together with standard deviation) in following table.

Table 13

Antibody	Cell factor stimulates	The +/- standard deviation of average pg/ml IFN-γs
			Without antibody	Nothing	87+/-7
Without antibody	1ng/ml IL-12	532+/-51
			Chimeric 18E5	1ng/ml IL-12	511+/-3
Chimeric 6B8	1ng/ml IL-12	523+/-60
			Antibody A	1ng/ml IL-12	497+/-30
Antibody B	1ng/ml IL-12	537+/-2
			Antibody C	1ng/ml IL-12	495+/-25
Antibody D	1ng/ml IL-12	539+/-38
			Anti- IL-12p70 antibody	1ng/ml IL-12	119+/-12

Embodiment 6：Suppress the STAT3 phosphorylations induced in Human cell line DB by IL-23

Human cell line DB (ATCC, Manassas, VA) passes through endogenous IL-23R compounds (IL-23R and IL-12R β 1) IL-23 is stimulated and produces response, and STAT3 phosphorylations are made with IL-23 dosage-dependent manners.Develop a kind of for testing Anti-il-23 p 19 antibodies suppress the analysis of the STAT3 phosphorylations of IL-23 inductions.DB cells are inoculated in 1 × 10e6 cells/well In 96 orifice plates.Pre-incubation 1 is small together with recombinant human IL-23 (10ng/ml) through serial dilution and in room temperature for the antibody to be tested When.Then at 37 DEG C, antibody/IL-23 mixtures is added in cell, continue 30 minutes.By centrifuging 10 points at 4 DEG C Clock collects cell and then in ice-cold buffer (Cell Signaling Technology, Beverly, MA) that cell is molten Solution.Phosphoric acid STAT3ELISA (Invitrogen) is carried out to a part of lysate.According to compared to without antibody control wells STAT3 inhibition of phosphorylation percentage calculates antibody I C₅₀Value.Representative IC₅₀Value is showed in following table.

Table 14

Antibody	IC₅₀(pM)
		Antibody A	25,15,38,23,13,18
Antibody B	73,84
		Antibody C	132,80
Antibody D	158
		QF20	26,26,27
C-1273	163,438

Embodiment 7：The vivo models for producing cell factor are induced in mouse ear by IL-23

Use the vivo models in mouse.Recombinant human IL-23 was injected in the skin of mouse ear in continuous 4 days, from And cause epidermis thickening and IL-17 and IL-22 protein upregulations.Anti-il-23 p 19 antibodies are assessed in this model.For the first time by IL- 23 are injected in skin 1 hour before, and 1mg/kg or 5mg/kg antibody are given through single intraperitoneal injection.Continue 3 days again often Day shot recombinant human IL-23 (it contains joint) and collection tissue progress cell factor assessment.Show antibody on cell Inhibitory action caused by the factor.The result of three experiments is showed in following table (experiment 1：1-7 rows, experiment 2：8-10 rows, it is real Test 3：1-14 rows).

Table 15

Embodiment 8：Pharmacokinetic study in stump-tailed macaque

Humanization anti-il-23 p 19 antibodies are given to three stump-tailed macaques by intravenous infusion ten minutes, dosage is 1.0mg/kg.Blood serum sample is collected after 6 weeks time-histories, and uses specific ELISA measurement free antibody concentrations.The serum of antibody is dense Degree-time graph and relative medicine kinetic parameter are summarized in table 16 below.

Table 16

Antibody	CL(ml/d/kg)	Vol(ml/kg)	AUC(nM·h/ml)	T_1/2(my god)	MRT (my god)
						Antibody A	5.2	88	32262	12.1	17.2
Antibody B	6.0	87	27030	10.1	14.8
						Antibody C	4.7	91	34642	14.1	19.6
Antibody D	3.4	67	47633	12.6	19.8

Embodiment 9：Expression and biophysical data in NSO cells

Transfect NS0 cells and produce stable cell pool (pool)：

NS0 cells is grown in the presence of 1%FBS, then transfected.Collect 40 × 10e6 cell, and with 20 μ g Linearisation DNA (heavy chain and light chain expression vector) is resuspended in the 0.8ml culture mediums containing 2%FBS together, then on ice Cell is cultivated about 15 minutes, then to cell electroporation under 750V/25 μ F (Bio-Rad Gene Pulser Xcell). Reclaim cell about 48 hours with 2%FBS under 37 DEG C and 5%CO2, be then inoculated in containing G418 with 2 × 10e5 cell/ml and In 96 orifice plates of mycophenolic acid, continue 14-21 days until forming group.

The supernatant of group is carried from the screening of 96 orifice plates by ELISA.Elisa plate, which is used, contains the 1 anti-κ of μ g/ml goats The PBS coatings of (Southern Biotech, Birmingham, AL), and diluted supernatant is cultivated, it is then anti-with goat IgG Fc-HRP (coming from Jackson ImmunoResearch Laboratories, West Grove, PA) is detected.Converge Heyang group is to expand scale.Albumin A tip (protein A tips) is used according to manufacturer's scheme, passes through ForteBio Determine potency caused by antibody.Antibody A and antibody D potency are between 250-350mg/L, from the rate of recovery of protein purification More than 80%, and containing the monomer more than 94% after IEX purifying.Protein is resuspended in containing 20mM sodium citrates and Stable at least four moon is kept in 115mM NaCl final buffer solution (pH 6.0) and at 4 DEG C, and it is molten in the buffer solution Xie Du is up to 100mg/ml.

Table 17

Table 18

AUC：Under 0.5-1mg/ml concentration, analytical ultracentrifugation is carried out according to the measurement of sedimentation velocity method；SEC：Chi Very little exclusion chromatography；%M：Monomer percentage.

Embodiment 10：Epitope mapping (mapping)

Mass spectrum (HXMS) is exchanged using hydrogen/deuterium to position the epitope that antibody A is combined with mankind IL-23p19.The method determines IL-23p19 amide backbone hydrogen and D₂The liability that O is exchanged.Experiment is with single IL-23 and the IL-23 added with antibody A Carry out.Thus identify IL-23p19 sequences in display because antibody A with reference to and significantly from the region of exchange.The resolution of method Rate depends on the peptide caused by pepsin or protease XVIII digestion.By using standard exact mass and HPLC MS/ MS technologies carry out other control experiments to identify these peptides for coming from IL-23p19 with sample is not exchanged.

Use recombinant human IL-23.For protein+antibody samples, 50 μ l IL-23 (0.8mg/ml) and 10 μ l antibody As (12.7mg/ml) is cultivated 15 minutes in room temperature together.Antibody A/IL-23 final mol ratio is 1.2:1.

To swap, 5 μ l IL-23 albumen are added into the 50 μ l deuterates buffer solution (D of the PBS containing 50mM₂O in) and in room Temperature is cultivated 100 seconds.Add 50 μ l 2M ureas/0.5M TCEP and cultivated 60 seconds in room temperature.Add 5 μ l pepsins or protease XVIII (4mg/ml, is dissolved in 0.1% formic acid), and sample is cooled to 4 DEG C at once.

After five minutes, by 50 μ l sample injections, in Shimadzu HPLC systems, (SCL10A is controlled under the following conditions Device and two LC10AD pumps) on：

Mobile phase A=99/1/0.1 (water/acetonitrile/formic acid).

Mobile phase B=95/5/0.1 (acetonitrile/water/formic acid).

The μ l/min of flow velocity=100.

Post=Phenomenex Jupiter C5,5 μ, 50 × 1.0mm.

Mobile phase pipeline, post, syringe ring are in ice bath.

Gradient=time 0 (3%B), time 2.2 (3%B), time 10.1 (90%B), time 12.0 (90%B), time 12.1 (3%B).

It is carried out as follows mass spectral analysis：

Mass spectrograph=Thermo Orbitrap Velos (0900865).

Method：

A. break (to identify peptide)：12 minutes acquisition times (start delay in 3 minutes), sweep entirely under 30,000 resolution ratio Retouch FTMS, seven secondary ion trap data dependencies scanning (CID).

B.MS is operated：12 minutes acquisition times (start delay in 3 minutes), the full scan FTMS under 60,000 resolution ratio.

Use break data and program Proteome Discoverer (Thermoscientific, Waltham, MA) Identify pepsin and protease XVIII peptides.The peptide identified is carried out using Xcalibur softwares (Thermoscientific) Visually relatively (single protein and the protein that antibody be present).For for single IL-23 and having the IL-23 of antibody A, Outside in IL-23p19 areas changes without significantly exchange.For the p19 parts of protein, program PepMap is used (Thermoscientific) analyze data.This program calculates the average quality for exchanging peptide.Check PepMap results and with Those peptides for not producing institute's the result are calculated in Microsoft Excel.

In IL-23 sequences display because antibody A with reference to and significantly from the region of exchange, be accredited as SEQID NO:181 Amino acid residue 108 to 126 and SEQ ID NO:181 amino acid residue 137 to 151.

The present invention relates to following items：

1. anti-il-23 p 19 antibodies or its antigen-binding fragment, the wherein antibody or its antigen-binding fragment include：

A) light chain variable district, the light chain variable district include amino acid sequence SEQ ID NO:19(CDR1-L)；Amino acid sequence Arrange SEQ ID NO:20(CDR2-L)；And amino acid sequence SEQ ID NO:21(CDR3-L)；And

B) weight chain variable district, the weight chain variable district include amino acid sequence SEQ ID NO:63rd, 66,67 or 68 (CDR1- H)；Amino acid sequence SEQ ID NO:64(CDR2-H)；And amino acid sequence SEQ ID NO:65(CDR3-H).

2. such as the anti-il-23 p 19 antibodies or its antigen-binding fragment of item 1, the wherein antibody or its antigen-binding fragment bag Contain：

B) weight chain variable district, the weight chain variable district include amino acid sequence SEQ ID NO:63(CDR1-H)；Amino acid sequence Arrange SEQ ID NO:64(CDR2-H)；And amino acid sequence SEQ ID NO:65(CDR3-H).

3. such as the anti-il-23 p 19 antibodies or its antigen-binding fragment of item 1, the wherein antibody or its antigen-binding fragment bag Contain：

B) weight chain variable district, the weight chain variable district include amino acid sequence SEQ ID NO:66(CDR1-H)；Amino acid sequence Arrange SEQ ID NO:64(CDR2-H)；And amino acid sequence SEQ ID NO:65(CDR3-H).

4. such as the anti-il-23 p 19 antibodies or its antigen-binding fragment of item 1, the wherein antibody or its antigen-binding fragment bag Contain：

B) weight chain variable district, the weight chain variable district include amino acid sequence SEQ ID NO:67(CDR1-H)；Amino acid sequence Arrange SEQ ID NO:64(CDR2-H)；And amino acid sequence SEQ ID NO:65(CDR3-H).

5. such as the anti-il-23 p 19 antibodies or its antigen-binding fragment of item 1, the wherein antibody or its antigen-binding fragment bag Contain：

B) weight chain variable district, the weight chain variable district include amino acid sequence SEQ ID NO:68(CDR1-H)；Amino acid sequence Arrange SEQ ID NO:64(CDR2-H)；And amino acid sequence SEQ ID NO:65(CDR3-H).

6. such as the anti-il-23 p 19 antibodies or its antigen-binding fragment of item 1, the wherein antibody or its antigen-binding fragment bag Contain：Light chain variable district, the light chain variable district include amino acid sequence SEQ ID NO:158th, any one of 160,162 or 164；And Weight chain variable district, the weight chain variable district include amino acid sequence SEQ ID NO:166th, any one of 168,170 or 172.

7. such as the anti-il-23 p 19 antibodies or its antigen-binding fragment of item 6, the wherein antibody or its antigen-binding fragment bag Containing containing amino acid sequence SEQ ID NO:160 light chain variable district and contain amino acid sequence SEQ ID NO:166 heavy chain Variable region.

8. such as the anti-il-23 p 19 antibodies or its antigen-binding fragment of item 6, the wherein antibody or its antigen-binding fragment bag Containing containing amino acid sequence SEQ ID NO:160 light chain variable district and contain amino acid sequence SEQ ID NO:168 heavy chain Variable region.

9. such as the anti-il-23 p 19 antibodies or its antigen-binding fragment of item 6, the wherein antibody or its antigen-binding fragment bag Containing containing amino acid sequence SEQ ID NO:158 light chain variable district and contain amino acid sequence SEQ ID NO:166 heavy chain Variable region.

10. such as the anti-il-23 p 19 antibodies or its antigen-binding fragment of item 6, the wherein antibody or its antigen-binding fragment bag Containing containing amino acid sequence SEQ ID NO:158 light chain variable district and contain amino acid sequence SEQ ID NO:168 heavy chain Variable region.

11. such as the anti-il-23 p 19 antibodies or its antigen-binding fragment of item 6, the wherein antibody or its antigen-binding fragment bag Containing the amino acid sequence SEQ ID NO being connected with the heavy chain constant region of IgG 1:166 or 168, and it is constant with human kappa light chain The amino acid sequence SEQ ID NO of area's connection:158 or 160.

12. such as the anti-il-23 p 19 antibodies or its antigen-binding fragment of item 1, the wherein antibody or its antigen-binding fragment bag Contain：

A) humanization light-chain variable domain, the light-chain variable domain include SEQ ID NO:158 or 160 CDR, and amino acid Sequence and SEQ ID NO:The skeleton region amino acid sequence at least 90% of 158 or 160 variable domain light-chain amino acid sequence is consistent Skeleton area；And

B) humanized heavy chain's variable domain, the heavy chain variable domain include SEQ ID NO:166 or 168 CDR, and amino acid Sequence and SEQ ID NO:The skeleton region amino acid sequence at least 90% of 166 or 168 variable domain heavy chain amino acid sequence is consistent Skeleton area.

13. such as any one of item 1-12 anti-il-23 p 19 antibodies, wherein the antibody is monoclonal antibody.

14. such as anti-il-23 p 19 antibodies of item 1, wherein the antibody is Humanized monoclonal anti-il-23 p 19 antibodies, and Comprising：Containing selected from SEQ ID NO:158th, the light chain variable district of 160,162 and 164 amino acid sequence of any one, and Containing selected from SEQ ID NO:166th, the weight chain variable district of 168,170 and 172 amino acid sequence of any one.

15. such as the Humanized monoclonal anti-il-23 p 19 antibodies of item 14, the wherein antibody contains amino acid sequence SEQ ID NO:160 light chain variable district and contain amino acid sequence SEQ ID NO:166 weight chain variable district.

16. such as the Humanized monoclonal anti-il-23 p 19 antibodies of item 14, the wherein antibody contains amino acid sequence SEQ ID NO:160 light chain variable district and contain amino acid sequence SEQ ID NO:168 weight chain variable district.

17. such as the Humanized monoclonal anti-il-23 p 19 antibodies of item 14, the wherein antibody contains amino acid sequence SEQ ID NO:158 light chain variable district and contain amino acid sequence SEQ ID NO:166 weight chain variable district.

18. such as the Humanized monoclonal anti-il-23 p 19 antibodies of item 14, the wherein antibody contains amino acid sequence SEQ ID NO:158 light chain variable district and contain amino acid sequence SEQ ID NO:168 weight chain variable district.

19. such as anti-il-23 p 19 antibodies of item 1, wherein the antibody is Humanized monoclonal anti-il-23 p 19 antibodies, its Comprising：Contain amino acid sequence SEQ ID NO:174 or 180 light chain and contain amino acid sequence SEQ ID NO:176 or 178 Heavy chain.

20. such as the Humanized monoclonal anti-il-23 p 19 antibodies of item 19, the wherein antibody contains amino acid sequence SEQ ID NO:174 light chain and contain amino acid sequence SEQ ID NO:176 heavy chain.

21. such as the Humanized monoclonal anti-il-23 p 19 antibodies of item 19, the wherein antibody contains amino acid sequence SEQ ID NO:174 light chain and contain amino acid sequence SEQ ID NO:178 heavy chain.

22. such as the Humanized monoclonal anti-il-23 p 19 antibodies of item 19, the wherein antibody contains amino acid sequence SEQ ID NO:180 light chain and contain amino acid sequence SEQ ID NO:176 heavy chain.

23. such as the Humanized monoclonal anti-il-23 p 19 antibodies of item 19, the wherein antibody contains amino acid sequence SEQ ID NO:180 light chain and contain amino acid sequence SEQ ID NO:178 heavy chain.

24. anti-il-23 p 19 antibodies or its antigen-binding fragment, it is by SEQ ID NO:181 amino acid residue 108 Combined at the epitope formed to 126 and amino acid residue 137 to 151 with mankind IL-23p19.

25. anti-il-23 p 19 antibodies or its antigen-binding fragment, itself and following antibody competition combination mankind IL-23p19：

A) Humanized monoclonal anti-il-23 p 19 antibodies, it contains amino acid sequence SEQ ID NO:174 light chain And contain amino acid sequence SEQ ID NO:176 heavy chain；Or

B) Humanized monoclonal anti-il-23 p 19 antibodies, it contains amino acid sequence SEQ ID NO:174 light chain And contain amino acid sequence SEQ ID NO:178 heavy chain；Or

C) Humanized monoclonal anti-il-23 p 19 antibodies, it contains amino acid sequence SEQ ID NO:180 light chain And contain amino acid sequence SEQ ID NO:176 heavy chain；Or

D) Humanized monoclonal anti-il-23 p 19 antibodies, it contains amino acid sequence SEQ ID NO:180 light chain And contain amino acid sequence SEQ ID NO:178 heavy chain.

26. such as anti-il-23 p 19 antibodies of any one of item 24 or 25, wherein the antibody is monoclonal antibody.

27. such as any one of item 1-26 antibody or antigen-binding fragment, it is used in medicine.

28. as item 27 antibody or antigen-binding fragment, wherein the purposes for treatment inflammatory disease, autoimmune disease, Respiratory disease, dysbolism or cancer.

29. such as the antibody or antigen-binding fragment of item 27, the wherein purposes is treatment psoriasis, inflammatory bowel disease, psoriasis Arthritis, multiple sclerosis, rheumatoid arthritis or ankylosing spondylitis.

30. pharmaceutical composition, it includes such as any one of item 1-26 antibody or antigen-binding fragment and pharmaceutically may be used The supporting agent of receiving.

31. individual method of the treatment with IL-23 associated conditions, it includes：Given to the individual as in item 1-26 The antibody or antigen-binding fragment of any one or the pharmaceutical composition such as item 30, the antibody or antigen-binding fragment and the mankind IL-23 is combined.

32. treating the method for inflammatory disease, autoimmune disease, respiratory disease, dysbolism or cancer, it includes The antibody or antigen-binding fragment such as any one of item 1-26 of effective dose or the medicine such as item 30 are given to individuals in need Compositions.

33. as item 32 method, wherein the disease be psoriasis, inflammatory bowel disease, psoriatic arthritis, multiple sclerosis, Rheumatoid arthritis or ankylosing spondylitis.

34. suppress the methods that are combined with the DCRS5 on mammalian cell of IL-23, it include to the cell to The antibody or antigen-binding fragment such as any one of item 1-26 are given, thus suppresses the signal transduction mediated by the DCRS5.

35. a kind of anti-il-23 p 19 antibodies or its antigen-binding fragment, it contains following light chain variable district：

A) light chain CDR1 (L-CDR1) sequence SEQ ID NO:1st, 4,6,7,8,11,15,18,19,22,27 or 30；

B) light chain CDR2 (L-CDR2) sequence SEQ ID NO:2nd, 5,9,12,16,20,23,25,28 or 31；And

C) light chain CDR3 (L-CDR3) sequence SEQ ID NO:3rd, 10,13,14,17,21,24,26,29 or 32；

And contain following weight chain variable district：

D) heavy chain CDR1 (H-CDR1) sequence SEQ ID NO:33、36、38、40、43、45、48、51、54、57、60、63、 66th, 67,68,69,77 or 80；

E) heavy chain CDR2 (H-CDR2) sequence SEQ ID NO:34、39、41、46、49、52、55、58、61、64、70、72、 73rd, 75,78 or 81；And

F) heavy chain CDR3 (H-CDR3) sequence SEQ ID NO:35、37、42、44、47、50、53、56、59、62、65、71、 74th, 76,79 or 82.

36. such as the anti-il-23 p379 antibody or its antigen-binding fragment of item 35, the wherein antibody or its antigen-binding fragment Comprising：

37. such as the anti-il-23 p389 antibody or its antigen-binding fragment of item 36, the wherein antibody or its antigen-binding fragment Comprising：

A) amino acid sequence SEQ ID NO are contained:84 light chain variable district and contain amino acid sequence SEQ ID NO:121 Weight chain variable district；Or

B) amino acid sequence SEQ ID NO are contained:86 light chain variable district and contain amino acid sequence SEQ ID NO:123 Weight chain variable district；Or

C) amino acid sequence SEQ ID NO are contained:88 light chain variable district and contain amino acid sequence SEQ ID NO:125 Weight chain variable district；Or

D) amino acid sequence SEQ ID NO are contained:90 light chain variable district and contain amino acid sequence SEQ ID NO:127 Weight chain variable district；Or

E) amino acid sequence SEQ ID NO are contained:91 light chain variable district and contain amino acid sequence SEQ ID NO:128 Weight chain variable district；Or

F) amino acid sequence SEQ ID NO are contained:93 light chain variable district and contain amino acid sequence SEQ ID NO:130 Weight chain variable district；Or

G) amino acid sequence SEQ ID NO are contained:95 light chain variable district and contain amino acid sequence SEQ ID NO:132 Weight chain variable district；Or

H) amino acid sequence SEQ ID NO are contained:97 light chain variable district and contain amino acid sequence SEQ ID NO:134 Weight chain variable district；Or

I) amino acid sequence SEQ ID NO are contained:99 light chain variable district and contain amino acid sequence SEQ ID NO:136 Weight chain variable district；Or

J) amino acid sequence SEQ ID NO are contained:101 light chain variable district and contain amino acid sequence SEQ ID NO: 138 weight chain variable district；Or

K) amino acid sequence SEQ ID NO are contained:103 light chain variable district and contain amino acid sequence SEQ ID NO: 140 weight chain variable district；Or

L) amino acid sequence SEQ ID NO are contained:105 light chain variable district and contain amino acid sequence SEQ ID NO: 142 weight chain variable district；Or

M) amino acid sequence SEQ ID NO are contained:107 light chain variable district and contain amino acid sequence SEQ ID NO: 144 weight chain variable district；Or

N) amino acid sequence SEQ ID NO are contained:109 light chain variable district and contain amino acid sequence SEQ ID NO: 146 weight chain variable district；Or

O) amino acid sequence SEQ ID NO are contained:111 light chain variable district and contain amino acid sequence SEQ ID NO: 148 weight chain variable district；Or

P) amino acid sequence SEQ ID NO are contained:113 light chain variable district and contain amino acid sequence SEQ ID NO: 150 weight chain variable district；Or

Q) amino acid sequence SEQ ID NO are contained:115 light chain variable district and contain amino acid sequence SEQ ID NO: 152 weight chain variable district；Or

R) amino acid sequence SEQ ID NO are contained:117 light chain variable district and contain amino acid sequence SEQ ID NO: 154 weight chain variable district；Or

S) amino acid sequence SEQ ID NO are contained:119 light chain variable district and contain amino acid sequence SEQ ID NO: 156 weight chain variable district.

38. the polynucleotide of separation, it is included：

The sequence of the light chain variable district of encoding antibody or antibody fragment, the light chain variable district have following amino acid sequence： SEQ ID NO:84th, 86,88,90,91,93,95,97,99,101,103,105,107,109,111,113,115,117 or 119；Or

The sequence of the weight chain variable district of encoding antibody or antibody fragment, the weight chain variable district have following amino acid sequence： SEQ ID NO:121、123、125、127、128、130、132、134、136、138、140、142、144、146、148、150、 152nd, 154 or 156.

39. the polynucleotide of separation, it is included：

The sequence of the light chain variable district of encoding antibody or antibody fragment, the light chain variable district have following amino acid sequence： SEQ ID NO:158th, 160,162 or 164；Or

The sequence of the weight chain variable district of encoding antibody or antibody fragment, the weight chain variable district have following amino acid sequence： SEQ ID NO:166th, 168,170 or 172.

40. the polynucleotide of the separation such as item 39, wherein the polynucleotide includes：The sequence of encoding antibody light, institute Stating light chain has amino acid sequence SEQ ID NO:174 or 180；Or the sequence of encoding antibody heavy, the heavy chain have amino Acid sequence SEQ ID NO:176 or 178.

Sequence table

<110> Barrett, Rachel Rebecca

Canada, Keith

Catron, Katrina Mary

Copenhaver, Robert

Frego, Lee Edward

Raymond, Ernest Lee

Singh, Sanjaya

Zhu, Xiangyang

<120>Anti-il-23 antibodies

<130> 09-0561

<150> 61/410,158

<151> 2010-11-04

<150> 61/411,953

<151> 2010-11-10

<150> 61/412,594

<151> 2010-11-11

<150> 61/448,785

<151> 2011-03-03

<160> 181

<170>FastSEQ, for Windows Version 4.0

<210> 1

<211> 11

<212> PRT

<213>Mouse (Mouse)

<400> 1

Arg Ala Ser Gln Ser Ile Ser Glu Tyr Leu His

1 5 10

<210> 2

<211> 7

<212> PRT

<213>Mouse (Mouse)

<400> 2

Tyr Ala Ser Gln Ser Ile Ser

1 5

<210> 3

<211> 9

<212> PRT

<213>Mouse (Mouse)

<400> 3

Gln Asn Gly His Ser Phe Pro Phe Thr

1 5

<210> 4

<211> 11

<212> PRT

<213>Mouse (Mouse)

<400> 4

Arg Ala Ser Gln Ser Ile Ser Asp Tyr Leu Tyr

1 5 10

<210> 5

<211> 7

<212> PRT

<213>Mouse (Mouse)

<400> 5

Phe Ala Ser Gln Ser Ile Ser

1 5

<210> 6

<211> 11

<212> PRT

<213>Mouse (Mouse)

<400> 6

Arg Ala Ser Gln Ser Ile Ser Val Tyr Leu His

1 5 10

<210> 7

<211> 11

<212> PRT

<213>Mouse (Mouse)

<400> 7

Arg Ala Ser Gln Ser Ile Ser Asp Tyr Leu His

1 5 10

<210> 8

<211> 16

<212> PRT

<213>Mouse (Mouse)

<400> 8

Arg Ser Ser Gln Ser Leu Val His Ser Asn Gly Asn Thr Tyr Leu His

1 5 10 15

<210> 9

<211> 7

<212> PRT

<213>Mouse (Mouse)

<400> 9

Lys Val Ser Asn Arg Phe Ser

1 5

<210> 10

<211> 9

<212> PRT

<213>Mouse (Mouse)

<400> 10

Ser Gln Ser Thr His Val Pro Tyr Thr

1 5

<210> 11

<211> 15

<212> PRT

<213>Mouse (Mouse)

<400> 11

Arg Ala Ser Lys Ser Val Ser Thr Ser Gly Tyr Ser Tyr Ile His

1 5 10 15

<210> 12

<211> 7

<212> PRT

<213>Mouse (Mouse)

<400> 12

Leu Ala Ser Asn Leu Asp Ser

1 5

<210> 13

<211> 9

<212> PRT

<213>Mouse (Mouse)

<400> 13

Gln His Ser Arg Glu Leu Pro Tyr Thr

1 5

<210> 14

<211> 9

<212> PRT

<213>Mouse (Mouse)

<400> 14

Gln Asn Gly His Ser Phe Pro Tyr Thr

1 5

<210> 15

<211> 15

<212> PRT

<213>Mouse (Mouse)

<400> 15

Arg Thr Ser Glu Ser Val Tyr Ser Tyr Gly Gln Asn Phe Ile His

1 5 10 15

<210> 16

<211> 7

<212> PRT

<213>Mouse (Mouse)

<400> 16

Arg Ala Ser Asn Leu Glu Ser

1 5

<210> 17

<211> 9

<212> PRT

<213>Mouse (Mouse)

<400> 17

Gln Gln Thr Asn Glu Asp Pro Tyr Thr

1 5

<210> 18

<211> 15

<212> PRT

<213>Mouse (Mouse)

<400> 18

Arg Ala Ser Glu Thr Ile Asn Phe Tyr Gly Thr Ser Phe Met His

1 5 10 15

<210> 19

<211> 11

<212> PRT

<213>Mouse (Mouse)

<400> 19

Lys Ala Ser Arg Asp Val Ala Ile Ala Val Ala

1 5 10

<210> 20

<211> 7

<212> PRT

<213>Mouse (Mouse)

<400> 20

Trp Ala Ser Thr Arg His Thr

1 5

<210> 21

<211> 9

<212> PRT

<213>Mouse (Mouse)

<400> 21

His Gln Tyr Ser Ser Tyr Pro Phe Thr

1 5

<210> 22

<211> 11

<212> PRT

<213>Mouse (Mouse)

<400> 22

Arg Ala Ser Glu Asn Ile Asp Ser Tyr Leu Ala

1 5 10

<210> 23

<211> 7

<212> PRT

<213>Mouse (Mouse)

<400> 23

Ala Ala Arg Asn Leu Ala Asp

1 5

<210> 24

<211> 9

<212> PRT

<213>Mouse (Mouse)

<400> 24

Gln His Tyr Tyr Ser Thr Pro Phe Thr

1 5

<210> 25

<211> 7

<212> PRT

<213>Mouse (Mouse)

<400> 25

Ala Ala Thr Asn Leu Ala Asp

1 5

<210> 26

<211> 9

<212> PRT

<213>Mouse (Mouse)

<400> 26

Leu His Tyr Tyr Ser Thr Pro Phe Thr

1 5

<210> 27

<211> 15

<212> PRT

<213>Mouse (Mouse)

<400> 27

Arg Ala Ser Lys Ser Val Arg Phe Ser Asp Tyr Phe Tyr Met His

1 5 10 15

<210> 28

<211> 7

<212> PRT

<213>Mouse (Mouse)

<400> 28

Leu Ala Ser Asn Leu Glu Ser

1 5

<210> 29

<211> 9

<212> PRT

<213>Mouse (Mouse)

<400> 29

Gln Asn Ser Arg Glu Leu Pro Tyr Thr

1 5

<210> 30

<211> 11

<212> PRT

<213>Mouse (Mouse)

<400> 30

Lys Ala Ser Gln Asp Val Gly Asn Ala Val Val

1 5 10

<210> 31

<211> 7

<212> PRT

<213>Mouse (Mouse)

<400> 31

Trp Ala Ser Thr Arg His Ile

1 5

<210> 32

<211> 8

<212> PRT

<213>Mouse (Mouse)

<400> 32

Gln Gln Tyr Ser Ser Tyr Leu Thr

1 5

<210> 33

<211> 10

<212> PRT

<213>Mouse (Mouse)

<400> 33

Gly Tyr Thr Phe Thr Ser Ser Val Ile His

1 5 10

<210> 34

<211> 17

<212> PRT

<213>Mouse (Mouse)

<400> 34

Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Lys Tyr Asn Glu Lys Phe Lys

1 5 10 15

Gly

<210> 35

<211> 6

<212> PRT

<213>Mouse (Mouse)

<400> 35

Arg Leu Asp Glu Ala Tyr

1 5

<210> 36

<211> 10

<212> PRT

<213>Mouse (Mouse)

<400> 36

Gly Tyr Thr Phe Thr Arg Tyr Leu Ile His

1 5 10

<210> 37

<211> 6

<212> PRT

<213>Mouse (Mouse)

<400> 37

Asn Trp Asp Leu Asp Tyr

1 5

<210> 38

<211> 10

<212> PRT

<213>Mouse (Mouse)

<400> 38

Gly Tyr Thr Phe Thr Ser Ser Val Met His

1 5 10

<210> 39

<211> 17

<212> PRT

<213>Mouse (Mouse)

<400> 39

Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Gln Tyr Asn Glu Lys Phe Lys

1 5 10 15

Gly

<210> 40

<211> 10

<212> PRT

<213>Mouse (Mouse)

<400> 40

Gly Tyr Thr Phe Thr Ser Ser Ile Ile His

1 5 10

<210> 41

<211> 17

<212> PRT

<213>Mouse (Mouse)

<400> 41

Tyr Ile Asn Pro Tyr Asp Asp Val Thr Lys Tyr Asn Glu Lys Phe Lys

1 5 10 15

Gly

<210> 42

<211> 6

<212> PRT

<213>Mouse (Mouse)

<400> 42

Arg Trp Asp Glu Ser Tyr

1 5

<210> 43

<211> 10

<212> PRT

<213>Mouse (Mouse)

<400> 43

Gly Tyr Thr Phe Thr Thr Ser Ile Met His

1 5 10

<210> 44

<211> 6

<212> PRT

<213>Mouse (Mouse)

<400> 44

Arg Trp Asp Glu Ala Tyr

1 5

<210> 45

<211> 10

<212> PRT

<213>Mouse (Mouse)

<400> 45

Gly Tyr Thr Phe Thr Asp Tyr Tyr Met Asn

1 5 10

<210> 46

<211> 17

<212> PRT

<213>Mouse (Mouse)

<400> 46

Val Ile Ile Pro Tyr Asn Gly Gly Thr Ser Tyr Asn Gln Lys Phe Lys

1 5 10 15

Gly

<210> 47

<211> 9

<212> PRT

<213>Mouse (Mouse)

<400> 47

Asp Gly His Arg Trp Tyr Phe Asp Val

1 5

<210> 48

<211> 10

<212> PRT

<213>Mouse (Mouse)

<400> 48

Gly Tyr Ser Phe Thr Gly Tyr Tyr Met Asn

1 5 10

<210> 49

<211> 17

<212> PRT

<213>Mouse (Mouse)

<400> 49

Glu Ile Ile Pro Thr Thr Gly Gly Thr Ser Tyr Asn Gln Lys Phe Lys

1 5 10 15

Ala

<210> 50

<211> 11

<212> PRT

<213>Mouse (Mouse)

<400> 50

Glu Ser Gly Gly Phe Tyr Trp Tyr Phe Asp Val

1 5 10

<210> 51

<211> 10

<212> PRT

<213>Mouse (Mouse)

<400> 51

Gly Tyr Thr Phe Thr Asp His Thr Ile His

1 5 10

<210> 52

<211> 17

<212> PRT

<213>Mouse (Mouse)

<400> 52

Tyr Ile Tyr Pro Arg Asp Gly Tyr Pro Lys Phe Asn Glu Lys Phe Lys

1 5 10 15

Gly

<210> 53

<211> 9

<212> PRT

<213>Mouse (Mouse)

<400> 53

Arg Pro Pro Tyr Tyr Ala Met Asp Tyr

1 5

<210> 54

<211> 10

<212> PRT

<213>Mouse (Mouse)

<400> 54

Gly Tyr Thr Phe Thr Arg Tyr Leu Met His

1 5 10

<210> 55

<211> 17

<212> PRT

<213>Mouse (Mouse)

<400> 55

Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Asn Tyr Asn Glu Lys Phe Lys

1 5 10 15

Gly

<210> 56

<211> 6

<212> PRT

<213>Mouse (Mouse)

<400> 56

Asn Trp Asp Tyr Ala Tyr

1 5

<210> 57

<211> 10

<212> PRT

<213>Mouse (Mouse)

<400> 57

Gly Phe Ser Leu Thr Thr Tyr Ala Ile Ser

1 5 10

<210> 58

<211> 16

<212> PRT

<213>Mouse (Mouse)

<400> 58

Val Ile Trp Thr Gly Gly Gly Thr Lys Tyr Asn Ser Ala Leu Lys Ser

1 5 10 15

<210> 59

<211> 11

<212> PRT

<213>Mouse (Mouse)

<400> 59

Lys Asp Tyr Asn Tyr Gly Gly Ala Met Asp Tyr

1 5 10

<210> 60

<211> 10

<212> PRT

<213>Mouse (Mouse)

<400> 60

Gly Phe Ser Leu Asn Asn Phe Ala Ile Ser

1 5 10

<210> 61

<211> 16

<212> PRT

<213>Mouse (Mouse)

<400> 61

Ala Ile Trp Thr Gly Gly Gly Thr Asn Tyr Asn Ser Ala Leu Lys Ser

1 5 10 15

<210> 62

<211> 11

<212> PRT

<213>Mouse (Mouse)

<400> 62

Lys Asp Tyr Ser Tyr Gly Gly Ala Met Asp Tyr

1 5 10

<210> 63

<211> 10

<212> PRT

<213>Mouse (Mouse)

<400> 63

Gly Asn Thr Phe Thr Asp Gln Thr Ile His

1 5 10

<210> 64

<211> 17

<212> PRT

<213>Mouse (Mouse)

<400> 64

Tyr Ile Tyr Pro Arg Asp Asp Ser Pro Lys Tyr Asn Glu Asn Phe Lys

1 5 10 15

Gly

<210> 65

<211> 11

<212> PRT

<213>Mouse (Mouse)

<400> 65

Pro Asp Arg Ser Gly Tyr Ala Trp Phe Ile Tyr

1 5 10

<210> 66

<211> 10

<212> PRT

<213>Artificial sequence

<220>

<223>Humanized sequence

<400> 66

Gly Tyr Thr Phe Thr Asp Gln Thr Ile His

1 5 10

<210> 67

<211> 10

<212> PRT

<213>Artificial sequence

<220>

<223>Humanized sequence

<400> 67

Gly Phe Thr Phe Thr Asp Gln Thr Ile His

1 5 10

<210> 68

<211> 10

<212> PRT

<213>Artificial sequence

<220>

<223>Humanized sequence

<400> 68

Gly Gly Thr Phe Thr Asp Gln Thr Ile His

1 5 10

<210> 69

<211> 10

<212> PRT

<213>Mouse (Mouse)

<400> 69

Gly Tyr Thr Phe Thr Arg Tyr Val Met His

1 5 10

<210> 70

<211> 17

<212> PRT

<213>Mouse (Mouse)

<400> 70

Tyr Ile Asn Pro Tyr Asn Asp Val Thr Lys Tyr Asn Glu Lys Phe Lys

1 5 10 15

Gly

<210> 71

<211> 6

<212> PRT

<213>Mouse (Mouse)

<400> 71

Asn Trp Asp Val Pro Tyr

1 5

<210> 72

<211> 17

<212> PRT

<213>Mouse (Mouse)

<400> 72

Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Lys Tyr Asn Glu Arg Phe Lys

1 5 10 15

Gly

<210> 73

<211> 17

<212> PRT

<213>Mouse (Mouse)

<400> 73

Val Ile Ile Pro Tyr Asn Gly Gly Ile Ser Tyr Asn Gln Lys Phe Lys

1 5 10 15

Gly

<210> 74

<211> 9

<212> PRT

<213>Mouse (Mouse)

<400> 74

Asn Asp Tyr Asp Trp Tyr Phe Asp Val

1 5

<210> 75

<211> 17

<212> PRT

<213>Mouse (Mouse)

<400> 75

Asp Phe Asn His Asn Asn Asp Val Ile Thr Tyr Asn Pro Lys Phe Lys

1 5 10 15

Gly

<210> 76

<211> 10

<212> PRT

<213>Mouse (Mouse)

<400> 76

Gly Leu Arg Gly Tyr Tyr Ala Met Asp Tyr

1 5 10

<210> 77

<211> 10

<212> PRT

<213>Mouse (Mouse)

<400> 77

Gly Tyr Ser Phe Thr Asp Tyr Asn Met Asn

1 5 10

<210> 78

<211> 17

<212> PRT

<213>Mouse (Mouse)

<400> 78

Val Ile Ile Pro Asn Tyr Gly Phe Thr Ser Tyr Asn Gln Asn Phe Lys

1 5 10 15

Gly

<210> 79

<211> 11

<212> PRT

<213>Mouse (Mouse)

<400> 79

Asp Gly Gly Ile Leu Leu Trp Tyr Leu Asp Val

1 5 10

<210> 80

<211> 10

<212> PRT

<213>Mouse (Mouse)

<400> 80

Gly Tyr Thr Phe Thr Asp Tyr Asn Met Asp

1 5 10

<210> 81

<211> 17

<212> PRT

<213>Mouse (Mouse)

<400> 81

Asp Ile Asn Pro His Asn Gly Gly Thr Ile Tyr Asn Gln Lys Phe Lys

1 5 10 15

Gly

<210> 82

<211> 13

<212> PRT

<213>Mouse (Mouse)

<400> 82

Asn Tyr Tyr Gly Ser Ser Tyr Gly Trp Tyr Phe Asp Val

1 5 10

<210> 83

<211> 333

<212> DNA

<213>Mouse (Mouse)

<400> 83

gacattgtgc tgacccaatc tccaggttct ttggctgtgt ctctagggca gagggccacc 60

atatcctgca gaaccagtga aagtgtttat agttatggcc aaaattttat acactggtac 120

cagcagaaac caggacagcc acccaaactc ctcatctatc gtgcatccaa cctggaatct 180

gggatccctg ccaggttcag tggcagtggg tctaggacag acttcaccct caccatgaat 240

cctgtggagg ctgatgatgt tgcaacctat tactgtcagc aaactaatga ggatccgtac 300

acgttcggag gggggaccaa gctggaaata aga 333

<210> 84

<211> 111

<212> PRT

<213>Mouse (Mouse)

<400> 84

Asp Ile Val Leu Thr Gln Ser Pro Gly Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Gln Arg Ala Thr Ile Ser Cys Arg Thr Ser Glu Ser Val Tyr Ser Tyr

20 25 30

Gly Gln Asn Phe Ile His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Arg Ala Ser Asn Leu Glu Ser Gly Ile Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr Met Asn

65 70 75 80

Pro Val Glu Ala Asp Asp Val Ala Thr Tyr Tyr Cys Gln Gln Thr Asn

85 90 95

Glu Asp Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Arg

100 105 110

<210> 85

<211> 321

<212> DNA

<213>Mouse (Mouse)

<400> 85

gacattgtga tgacccagtc tcacaaattc ttgtccacat cagtgggaga cagggtcacc 60

atcacttgca aggccagtcg ggatgtggct attgctgtag cctggtatca acagaaacca 120

gggcaatctc ctaaactact tcttttctgg gcatccaccc gacacactgg ggtccctgat 180

cgcttcacag gcagtggatc tcggacagat ttcactctca ccattagcaa tgtgcagtct 240

gaagacttgg cagattattt ctgtcaccaa tatagcagct atccattcac gttcggctcg 300

gggacaaagt tggaaataaa g 321

<210> 86

<211> 107

<212> PRT

<213>Mouse (Mouse)

<400> 86

Asp Ile Val Met Thr Gln Ser His Lys Phe Leu Ser Thr Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Arg Asp Val Ala Ile Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Leu

35 40 45

Phe Trp Ala Ser Thr Arg His Thr Gly Val Pro Asp Arg Phe Thr Gly

50 55 60

Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr Ile Ser Asn Val Gln Ser

65 70 75 80

Glu Asp Leu Ala Asp Tyr Phe Cys His Gln Tyr Ser Ser Tyr Pro Phe

85 90 95

Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 87

<211> 333

<212> DNA

<213>Mouse (Mouse)

<400> 87

gacattgcgc tgacccaatc tccagcttct ttggctgtgt ctctggggca gagggccacc 60

atatcctgca gagccagtga aactattaat ttttatggca ctagttttat gcactggtac 120

cagcagaaac caggacagtc acccaaactc ctcatctatc gtgcatccaa cctagaatct 180

gggatccctg ccaggttcag tggcagtggg tctaggacag acttcaccct caccattaat 240

cctgtggagg ctgatgatgt tgcaacctat tactgtcagc aaactaatga ggatccgtac 300

acgttcggag gggggactaa gttggaaata aaa 333

<210> 88

<211> 111

<212> PRT

<213>Mouse (Mouse)

<400> 88

Asp Ile Ala Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Thr Ile Asn Phe Tyr

20 25 30

Gly Thr Ser Phe Met His Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro

35 40 45

Lys Leu Leu Ile Tyr Arg Ala Ser Asn Leu Glu Ser Gly Ile Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr Ile Asn

65 70 75 80

Pro Val Glu Ala Asp Asp Val Ala Thr Tyr Tyr Cys Gln Gln Thr Asn

85 90 95

Glu Asp Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 89

<211> 336

<212> DNA

<213>Mouse (Mouse)

<400> 89

gatgttgtga tgacccaaac tccactctcc ctgcctgtca gtcttggaga tcaagcctcc 60

atctcttgca gatctagtca gagccttgta cacagtaatg gaaacaccta tttacattgg 120

tacctgcaga agccaggcca gtctccaaag ctcctgatct acaaagtttc caaccgattt 180

tctggggtcc cagacaggtt cagtggcagt ggatcaggga cagatttcac actcaagatc 240

aacagagtgg aggctgagga tctgggagtt tatttctgct ctcaaagtac acatgttccg 300

tacacgttcg gaggggggac ccagctggaa ataaaa 336

<210> 90

<211> 112

<212> PRT

<213>Mouse (Mouse)

<400> 90

Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly

1 5 10 15

Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser

20 25 30

Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Asn Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Ser

85 90 95

Thr His Val Pro Tyr Thr Phe Gly Gly Gly Thr Gln Leu Glu Ile Lys

100 105 110

<210> 91

<211> 107

<212> PRT

<213>Mouse (Mouse)

<400> 91

Asp Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Thr Pro Gly

1 5 10 15

Asp Arg Val Ser Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Asp Tyr

20 25 30

Leu His Trp Tyr Gln Gln Arg Ser His Glu Ser Pro Arg Leu Leu Ile

35 40 45

Lys Tyr Ala Ser Gln Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Ser Asp Phe Thr Leu Thr Ile Asn Ser Val Glu Pro

65 70 75 80

Glu Asp Val Gly Val Tyr Tyr Cys Gln Asn Gly His Ser Phe Pro Phe

85 90 95

Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 92

<211> 321

<212> DNA

<213>Mouse (Mouse)

<400> 92

gacattgtga tgactcagtc tccagccacc ctgtctgtga ctccaggaga tagagtctct 60

ctttcctgca gggccagtca gagtattagc gactacttat actggtatca acaaaaatca 120

catgagtctc caaggcttct catcaaattt gcttcccaat ccatctctgg gatcccctcc 180

aggttcactg gcagtggatc agggtcagat ttcactctca gtatcgacag tgtggaacct 240

gatgatgttg gagtcttttt ctgtcaaaat ggtcacagct ttccgttcac gttcggaggg 300

gggaccaagc tggaaataaa a 321

<210> 93

<211> 107

<212> PRT

<213>Mouse (Mouse)

<400> 93

Asp Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Thr Pro Gly

1 5 10 15

Asp Arg Val Ser Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Asp Tyr

20 25 30

Leu Tyr Trp Tyr Gln Gln Lys Ser His Glu Ser Pro Arg Leu Leu Ile

35 40 45

Lys Phe Ala Ser Gln Ser Ile Ser Gly Ile Pro Ser Arg Phe Thr Gly

50 55 60

Ser Gly Ser Gly Ser Asp Phe Thr Leu Ser Ile Asp Ser Val Glu Pro

65 70 75 80

Asp Asp Val Gly Val Phe Phe Cys Gln Asn Gly His Ser Phe Pro Phe

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 94

<211> 321

<212> DNA

<213>Mouse (Mouse)

<400> 94

gacattgtga tgactcagtc tccagccacc ctgtctgtga ctccaggaga tagagtctct 60

ctttcctgca gggccagcca gagtattagc gagtacttac actggtatca acaaaaatca 120

catgagtctc caaggcttct catcaaatat gcttcccaat ccatctctgg gatcccctcc 180

aggttcagtg gcagtggatc agggtcagat ttcactctca gtatcaacag tgtggaacct 240

gaagatgttg gagtgtatta ctgtcaaaat ggtcacagct ttccattcac gttcggctcg 300

gggacaaagt tggaaataaa a 321

<210> 95

<211> 107

<212> PRT

<213>Mouse (Mouse)

<400> 95

Asp Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Thr Pro Gly

1 5 10 15

Asp Arg Val Ser Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Glu Tyr

20 25 30

Leu His Trp Tyr Gln Gln Lys Ser His Glu Ser Pro Arg Leu Leu Ile

35 40 45

Lys Tyr Ala Ser Gln Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Ser Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Pro

65 70 75 80

Glu Asp Val Gly Val Tyr Tyr Cys Gln Asn Gly His Ser Phe Pro Phe

85 90 95

Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 96

<211> 321

<212> DNA

<213>Mouse (Mouse)

<400> 96

gacattgtga tgactcagtc tccagccacc ctgtctgtga ctccaggaga tagagtctct 60

ctttcctgca gggccagcca gagtattagc gactacttat actggtatca acaaaaatca 120

catgagtctc caaggcttct catcaaattt gcttcccaat ccatctctgg gatcccctcc 180

aggttcactg gcagtggatc agggtcagat ttcactctca gtatcgacag tgtggaacct 240

gatgatgttg gagtcttttt ctgtcaaaat ggtcacagct ttccgttcac gttcggaggg 300

gggaccaagc tggaaataaa a 321

<210> 97

<211> 107

<212> PRT

<213>Mouse (Mouse)

<400> 97

Asp Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Thr Pro Gly

1 5 10 15

Asp Arg Val Ser Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Asp Tyr

20 25 30

Leu Tyr Trp Tyr Gln Gln Lys Ser His Glu Ser Pro Arg Leu Leu Ile

35 40 45

Lys Phe Ala Ser Gln Ser Ile Ser Gly Ile Pro Ser Arg Phe Thr Gly

50 55 60

Ser Gly Ser Gly Ser Asp Phe Thr Leu Ser Ile Asp Ser Val Glu Pro

65 70 75 80

Asp Asp Val Gly Val Phe Phe Cys Gln Asn Gly His Ser Phe Pro Phe

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 98

<211> 321

<212> DNA

<213>Mouse (Mouse)

<400> 98

gacattgtga tgactcagtc tccagccacc ctgtctgtga ctccaggaga tagagtctct 60

ctttcctgca gggccagcca gagtattagc gagtatttac actggtatca acaaaaatca 120

catgagtctc caaggcttct catcaaatat gcttcccaat ccatctctgg gatcccctcc 180

aggttcagtg gcagtggatc agggtcagat ttcactctca gtatcaacag tgtggaacct 240

gaagatgttg gagtttatta ctgtcaaaat ggtcacagct ttccattcac gttcggctcg 300

gggacaaagt tggaaataaa a 321

<210> 99

<211> 107

<212> PRT

<213>Mouse (Mouse)

<400> 99

Asp Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Thr Pro Gly

1 5 10 15

Asp Arg Val Ser Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Glu Tyr

20 25 30

Leu His Trp Tyr Gln Gln Lys Ser His Glu Ser Pro Arg Leu Leu Ile

35 40 45

Lys Tyr Ala Ser Gln Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Ser Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Pro

65 70 75 80

Glu Asp Val Gly Val Tyr Tyr Cys Gln Asn Gly His Ser Phe Pro Phe

85 90 95

Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 100

<211> 321

<212> DNA

<213>Mouse (Mouse)

<400> 100

gacattgtga tgactcagtc tccagccacc ctgtctgtga ctccaggaga tagagtctct 60

ctctcctgca gggccagcca gagtattagc gtctacttac actggtatca acaaaaatca 120

cctgagtctc caaggcttct catcaaatat gcttcccaat ccatctctgg gatcccctcc 180

aggttcagtg gcagtggatc agggtcagat ttcactctca gtatcaacag tgtggaacct 240

gaagatgttg gagtttatta ctgtcaaaat ggtcacagct ttccattcac gttcggctcg 300

gggacaaagt tggaaataaa a 321

<210> 101

<211> 107

<212> PRT

<213>Mouse (Mouse)

<400> 101

Asp Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Thr Pro Gly

1 5 10 15

Asp Arg Val Ser Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Val Tyr

20 25 30

Leu His Trp Tyr Gln Gln Lys Ser Pro Glu Ser Pro Arg Leu Leu Ile

35 40 45

Lys Tyr Ala Ser Gln Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Ser Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Pro

65 70 75 80

Glu Asp Val Gly Val Tyr Tyr Cys Gln Asn Gly His Ser Phe Pro Phe

85 90 95

Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 102

<211> 321

<212> DNA

<213>Mouse (Mouse)

<400> 102

gacattgtga tgactcagtc tccagccacc ctgtctgtga ctccaggaaa tagagtctct 60

ctttcctgca gggccagcca gagtattagc gactacttac actggtatca acaaaaatca 120

catgagtctc caaggcttct catcaaatat gcttcccaat ccatctctgg gatcccctcc 180

aggttcagtg gcagtggatc agggtcaaat ttcactctca gtatcaacag tgtggaacct 240

gaagatgttg gagtgtatta ttgtcaaaat ggtcacagct ttccattcac gttcggctcg 300

gggacaaagt tggaaataaa a 321

<210> 103

<211> 107

<212> PRT

<213>Mouse (Mouse)

<400> 103

Asp Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Thr Pro Gly

1 5 10 15

Asn Arg Val Ser Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Asp Tyr

20 25 30

Leu His Trp Tyr Gln Gln Lys Ser His Glu Ser Pro Arg Leu Leu Ile

35 40 45

Lys Tyr Ala Ser Gln Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Ser Asn Phe Thr Leu Ser Ile Asn Ser Val Glu Pro

65 70 75 80

Glu Asp Val Gly Val Tyr Tyr Cys Gln Asn Gly His Ser Phe Pro Phe

85 90 95

Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 104

<211> 333

<212> DNA

<213>Mouse (Mouse)

<400> 104

gacattgtgc tgacacagtc tcctgcttcc ttagctgttt ctctggggca gagggccacc 60

atctcatgca gggccagcaa aagtgtcaga ttctctgact atttttatat gcactggtac 120

caacagaaac caggacagcc acccaaactc ctcatctacc ttgcatccaa cctagaatct 180

ggggtccctg ccaggttcag tggcagtggg tctgggacag acttcaccct caacatccat 240

cctgtggagg aggaggatgc tgcaacctat tactgtcaga acagtaggga gcttccgtac 300

acgttcggag gggggaccaa gctggagata aaa 333

<210> 105

<211> 111

<212> PRT

<213>Mouse (Mouse)

<400> 105

Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Lys Ser Val Arg Phe Ser

20 25 30

Asp Tyr Phe Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His

65 70 75 80

Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Asn Ser Arg

85 90 95

Glu Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 106

<211> 333

<212> DNA

<213>Mouse (Mouse)

<400> 106

gacattgtgt tgacacagtc tcctgcttcc ttagctgtat ctctggggca gagggccacc 60

atctcatgca gggccagcaa aagtgtcagt acatctggct atagttatat acactggtac 120

caacagaaac cgggacagcc acccaaattc ctcatctatc ttgcatccaa cctagattct 180

ggggtccctg ccaggttcag tggcagtggg tctgggacag acttcaccct caacatccat 240

cctgtggagg aggaggatgc tgcaacctat tactgtcagc acagtaggga gcttccgtac 300

acgttcggag gggggaccaa gctggaaata aaa 333

<210> 107

<211> 111

<212> PRT

<213>Mouse (Mouse)

<400> 107

Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Lys Ser Val Ser Thr Ser

20 25 30

Gly Tyr Ser Tyr Ile His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Phe Leu Ile Tyr Leu Ala Ser Asn Leu Asp Ser Gly Val Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His

65 70 75 80

Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln His Ser Arg

85 90 95

Glu Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 108

<211> 321

<212> DNA

<213>Mouse (Mouse)

<400> 108

gacattgtga tgactcagtc tccagccacc ctgtctgtga ctccaggaga tagagtctct 60

ctttcctgca gggccagcca gagtattagc gactacttac actggtatca acaaaaatca 120

catgagtctc caaggcttct catcaaatat gcttcccaat ccatctctgg gatcccctcc 180

aggttcagtg gcagtggatc agggtcagat ttcactctca gtatcaacag tgtggaacct 240

gaagatgttg gagtgtatta ctgtcaaaat ggtcacagct ttccgtacac gttcggaggg 300

gggaccaagc tggaaataaa a 321

<210> 109

<211> 107

<212> PRT

<213>Mouse (Mouse)

<400> 109

Asp Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Thr Pro Gly

1 5 10 15

Asp Arg Val Ser Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Asp Tyr

20 25 30

Leu His Trp Tyr Gln Gln Lys Ser His Glu Ser Pro Arg Leu Leu Ile

35 40 45

Lys Tyr Ala Ser Gln Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Ser Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Pro

65 70 75 80

Glu Asp Val Gly Val Tyr Tyr Cys Gln Asn Gly His Ser Phe Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 110

<211> 336

<212> DNA

<213>Mouse (Mouse)

<400> 110

gatgttgtga tgacccaaac tccactctcc ctgcctgtca gtcttggaga tcaagcctcc 60

atctcttgca gatctagtca gagccttgta cacagtaatg gaaacaccta tttacattgg 120

tacctgcaga agccaggcca gtctccaaag ctcctgatct acaaagtttc caaccgattt 180

tctggggtcc ccgacaggtt cagtggcagt ggatcaggga cagatttcac actcaagatc 240

agcagagtgg aggctgagga tctgggagtt tatttctgct ctcaaagtac acatgttccg 300

tacacgttcg gaggggggac caagctggaa ataaat 336

<210> 111

<211> 112

<212> PRT

<213>Mouse (Mouse)

<400> 111

Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly

1 5 10 15

Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser

20 25 30

Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Ser

85 90 95

Thr His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Asn

100 105 110

<210> 112

<211> 318

<212> DNA

<213>Mouse (Mouse)

<400> 112

gacattatga tgacccagtc tcacaaattc atgtccacat cagtaggaga cagggtcagc 60

atcacctgca aggccagtca ggatgtgggt aatgctgtgg tctggtatca acaaaaacca 120

gggcaatctc ctaaactact gatttactgg gcatccaccc ggcacattgg agtccctgat 180

cgcttcacag gcagtggatc tgggacagat ttcactctca ccattaccaa tgtgcagtct 240

gaagacttgg cagattattt ctgtcagcaa tatagcagct atctcacgtt cggtgctggg 300

accaagctgg agctgaaa 318

<210> 113

<211> 106

<212> PRT

<213>Mouse (Mouse)

<400> 113

Asp Ile Met Met Thr Gln Ser His Lys Phe Met Ser Thr Ser Val Gly

1 5 10 15

Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Gly Asn Ala

20 25 30

Val Val Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile

35 40 45

Tyr Trp Ala Ser Thr Arg His Ile Gly Val Pro Asp Arg Phe Thr Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Thr Asn Val Gln Ser

65 70 75 80

Glu Asp Leu Ala Asp Tyr Phe Cys Gln Gln Tyr Ser Ser Tyr Leu Thr

85 90 95

Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

100 105

<210> 114

<211> 336

<212> DNA

<213>Mouse (Mouse)

<400> 114

gatgttgtga tgacccaatc tccactctcc ctgcctgtca gtcttggaga tcaagcctcc 60

atctcttgca gatctagtca gagccttgta cacagtaatg gaaacaccta tctacattgg 120

tacctgctga agccaggcca gtctccaaag ctcctgatct acaaagtttc caaccgattt 180

tctggggtcc cagacaggtt cagtggcagt ggatcaggga cagatttcac actcaagatc 240

agcagagtgg aggctgagga tctgggagtt tatttctgct ctcaaagtac acatgttccg 300

tacacgttcg gaggggggac caagctggaa ataaaa 336

<210> 115

<211> 112

<212> PRT

<213>Mouse (Mouse)

<400> 115

Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Ser Leu Gly

1 5 10 15

Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser

20 25 30

Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Leu Lys Pro Gly Gln Ser

35 40 45

Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Ser

85 90 95

Thr His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 116

<211> 321

<212> DNA

<213>Mouse (Mouse)

<400> 116

gacatccaga tgactcagtc tccagttttc ctgtctgcat ctgtgggaga aactgtcacc 60

atcacatgtc gagcaagtga gaatattgac agttatttag catggtatca gcagaaacag 120

ggaaaatctc ctcagctcct ggtctttgct gcacgaaact tagcagatgg tgtgccatca 180

aggttcagtg gcagtggatc aggcacacag tattctctca agatcaacag aatgcagtct 240

gaagatgttg cgagatacta ctgtcaacat tattatagta ctccattcac gttcggctcg 300

gggacaaagt tggaaataga a 321

<210> 117

<211> 107

<212> PRT

<213>Mouse (Mouse)

<400> 117

Asp Ile Gln Met Thr Gln Ser Pro Val Phe Leu Ser Ala Ser Val Gly

1 5 10 15

Glu Thr Val Thr Ile Thr Cys Arg Ala Ser Glu Asn Ile Asp Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Val

35 40 45

Phe Ala Ala Arg Asn Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Gln Tyr Ser Leu Lys Ile Asn Arg Met Gln Ser

65 70 75 80

Glu Asp Val Ala Arg Tyr Tyr Cys Gln His Tyr Tyr Ser Thr Pro Phe

85 90 95

Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Glu

100 105

<210> 118

<211> 321

<212> DNA

<213>Mouse (Mouse)

<400> 118

gacatccaga tgactcagtc gccagcttcc ctgtctgcat ctgtgggaga aactgtcatc 60

ttcacatgtc gagcaagtga gaatattgac agttatttag catggtatca gcagaaacag 120

ggaaaatctc ctcagctcct ggtctatgct gcaacaaact tagcagatgg tgtgccatca 180

aggttcagtg gcagtggatc aggcacacag tattctctca agatcaacag cctgcagtct 240

gaagatgttg cgagatatta ctgtctacat tattatagta ctccattcac gttcggctcg 300

gggacagagt tggaaataaa a 321

<210> 119

<211> 107

<212> PRT

<213>Mouse (Mouse)

<400> 119

Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Glu Thr Val Ile Phe Thr Cys Arg Ala Ser Glu Asn Ile Asp Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Val

35 40 45

Tyr Ala Ala Thr Asn Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Gln Tyr Ser Leu Lys Ile Asn Ser Leu Gln Ser

65 70 75 80

Glu Asp Val Ala Arg Tyr Tyr Cys Leu His Tyr Tyr Ser Thr Pro Phe

85 90 95

Thr Phe Gly Ser Gly Thr Glu Leu Glu Ile Lys

100 105

<210> 120

<211> 357

<212> DNA

<213>Mouse (Mouse)

<400> 120

caggtgcagc tgaaggagtc aggacctggc ctggtggcgc cctcacagag cctgtccatc 60

acatgcactg tctctgggtt ctcattaacc acctatgcta taagctgggt tcgccagtca 120

ccaggaaagg gtctggagtg gcttggagtc atatggactg gtggaggcac aaaatataat 180

tcagctctca aatccagact gagcatcagc aaagacaact ccaagagtca agttttctta 240

aaaatgaaca gtctgcaaac tgatgacaca gccaggtact actgtgccag aaaggactat 300

aattacgggg gtgctatgga ctactggggt caaggaacct cagtcaccgt ctcctca 357

<210> 121

<211> 119

<212> PRT

<213>Mouse (Mouse)

<400> 121

Gln Val Gln Leu Lys Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln

1 5 10 15

Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Thr Tyr

20 25 30

Ala Ile Ser Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Gly Val Ile Trp Thr Gly Gly Gly Thr Lys Tyr Asn Ser Ala Leu Lys

50 55 60

Ser Arg Leu Ser Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Phe Leu

65 70 75 80

Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Arg Tyr Tyr Cys Ala

85 90 95

Arg Lys Asp Tyr Asn Tyr Gly Gly Ala Met Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Ser Val Thr Val Ser Ser

115

<210> 122

<211> 360

<212> DNA

<213>Mouse (Mouse)

<400> 122

caggttcagc tgcaacagtc tgacgctgag ttggtgaaac ctggcacttc agtgaagaca 60

tcctgcaaaa tttctggcaa caccttcact gaccaaacta ttcactggat gaagcagagg 120

cctgaacagg gcctggaatg gattggatat atttatccta gagatgatag tcctaagtac 180

aatgagaact tcaagggcaa ggccacattg actgcagaca aatcctccag cacagcctac 240

atgcagctca acagtctgac atctgaggac tctgcagtct atttctgtgc aatcccagac 300

aggtcaggct acgcctggtt tatttactgg ggccaaggga ctctggtcac tgtctcttca 360

<210> 123

<211> 120

<212> PRT

<213>Mouse (Mouse)

<400> 123

Gln Val Gln Leu Gln Gln Ser Asp Ala Glu Leu Val Lys Pro Gly Thr

1 5 10 15

Ser Val Lys Thr Ser Cys Lys Ile Ser Gly Asn Thr Phe Thr Asp Gln

20 25 30

Thr Ile His Trp Met Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Tyr Pro Arg Asp Asp Ser Pro Lys Tyr Asn Glu Asn Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys

85 90 95

Ala Ile Pro Asp Arg Ser Gly Tyr Ala Trp Phe Ile Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 124

<211> 357

<212> DNA

<213>Mouse (Mouse)

<400> 124

caggtgcagc tgaaggagtc aggacctgtc ctggtggcgc cctcacagag cctgtccatc 60

acatgcactg tctctgggtt ctcattaaac aactttgcta taagttgggt tcgtcagcca 120

ccaggaaagg gtctggagtg gcttggagca atatggactg gtggaggcac aaattataat 180

tcagctctca aatccagact gagcatcagc aaagacaact ccaagagtca agttttctta 240

aaaatgaaca gtctgcaaac tgatgacaca gccaggtatt attgtgtcag aaaggactat 300

agttacgggg gtgctatgga ctactggggt caaggaacct cagtcaccgt ctcctca 357

<210> 125

<211> 119

<212> PRT

<213>Mouse (Mouse)

<400> 125

Gln Val Gln Leu Lys Glu Ser Gly Pro Val Leu Val Ala Pro Ser Gln

1 5 10 15

Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Asn Asn Phe

20 25 30

Ala Ile Ser Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Gly Ala Ile Trp Thr Gly Gly Gly Thr Asn Tyr Asn Ser Ala Leu Lys

50 55 60

Ser Arg Leu Ser Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Phe Leu

65 70 75 80

Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Arg Tyr Tyr Cys Val

85 90 95

Arg Lys Asp Tyr Ser Tyr Gly Gly Ala Met Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Ser Val Thr Val Ser Ser

115

<210> 126

<211> 354

<212> DNA

<213>Mouse (Mouse)

<400> 126

gaggtccagc tgcaacagtc tggacctgtg ctggtgaagc ctggggcttc agtgaagatg 60

tcctgtaagg cttctggata cacattcact gactactata tgaactgggt gaagcagagc 120

catggaaaga gccttgagtg gattggagtt attattcctt acaacggtgg tactagctac 180

aaccagaagt tcaagggcaa ggccacattg actgttgaca agtcctccag cacagcctac 240

atggagctca acagcctgac atctgaggac tctgcagtct attactgtgc acgagatggt 300

caccgctggt acttcgatgt ctggggcaca gggaccacgg tcaccgtctc ctca 354

<210> 127

<211> 118

<212> PRT

<213>Mouse (Mouse)

<400> 127

Glu Val Gln Leu Gln Gln Ser Gly Pro Val Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Tyr Met Asn Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile

35 40 45

Gly Val Ile Ile Pro Tyr Asn Gly Gly Thr Ser Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Gly His Arg Trp Tyr Phe Asp Val Trp Gly Thr Gly Thr

100 105 110

Thr Val Thr Val Ser Ser

115

<210> 128

<211> 115

<212> PRT

<213>Mouse (Mouse)

<400> 128

Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Cys Cys

20 25 30

Ile Met His Trp Val Lys Gln Lys Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Lys Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Ser Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Trp Asp Glu Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ala

115

<210> 129

<211> 345

<212> DNA

<213>Mouse (Mouse)

<400> 129

gaggtccagc tgcagcagtc tggacctgag ctggtcaagc ctggggcttc agtgaagatg 60

tcctgcaagg cttctggata cacattcact cgctatctta ttcactgggt gaaacagaag 120

cctgggcagg gccttgagtg gattggatat attaatcctt acaatgatgg tactaaatac 180

aatgagaagt tcaaaggcaa ggccacactg acttcagaca aatcctccag cacagcctac 240

atggagctca gcagcctgac ctctgaggac tctgcggtct attactgtac ctctaactgg 300

gacctcgact actggggcca aggcaccact ctcacagtct cctca 345

<210> 130

<211> 115

<212> PRT

<213>Mouse (Mouse)

<400> 130

Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Arg Tyr

20 25 30

Leu Ile His Trp Val Lys Gln Lys Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Lys Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Ser Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Thr Ser Asn Trp Asp Leu Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr

100 105 110

Val Ser Ser

115

<210> 131

<211> 345

<212> DNA

<213>Mouse (Mouse)

<400> 131

gaggtccagc tgcagcagtc tggacctgaa gtggtaaagc ctggggcttc agtgaagatg 60

tcctgcaagg cctctggata cacattcact agttctgtta tacactgggt gaagcagaag 120

gctgggcagg gccttgagtg gattggatat atcaatccct ataatgatgg tactaagtac 180

aatgagaagt tcaaaggcaa ggccacactg acttcagaca gatcctccag cacagcctac 240

atggagctca gcagcctgac ctctgaggac tctgcggtct attactgtac aagacggttg 300

gacgaggctt actggggcca agggactctg gtcactgtct ctgca 345

<210> 132

<211> 115

<212> PRT

<213>Mouse (Mouse)

<400> 132

Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Val Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Ser

20 25 30

Val Ile His Trp Val Lys Gln Lys Ala Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Lys Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Ser Asp Arg Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Arg Leu Asp Glu Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ala

115

<210> 133

<211> 345

<212> DNA

<213>Mouse (Mouse)

<400> 133

gaggtccagc tgcagcagtc tggacctgag ctggtaaagc ctggggctgc agtgaagatg 60

tcctgcaagg cttctggata cacattcact cgctatctta ttcactgggt gaagcagaag 120

cctgggcagg gccttgagtg gattggatat attaatcctt acaatgatgg tactaaatat 180

aatgagaagt tcaaaggcaa ggccacactg acttcagaca aatcctccag cacagcctac 240

atggagctca gcagcctgac ctctgaggac tctgcggtct attactgtac ctctaattgg 300

gacctcgact actggggcca aggcaccact ctcacagtct cctca 345

<210> 134

<211> 115

<212> PRT

<213>Mouse (Mouse)

<400> 134

Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ala Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Arg Tyr

20 25 30

Leu Ile His Trp Val Lys Gln Lys Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Lys Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Ser Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Thr Ser Asn Trp Asp Leu Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr

100 105 110

Val Ser Ser

115

<210> 135

<211> 345

<212> DNA

<213>Mouse (Mouse)

<400> 135

gaggtccagc tgcagcagtc tggacctgaa ctggtaaagc ctggggcttc agtgaagatg 60

tcctgcaagg cctctggata cacattcact agttctgtta tgcactgggt gaagcagaag 120

gctgggcagg gccttgagtg gattggatat atcaatccct ataatgatgg tactcagtac 180

aatgagaagt tcaaaggcaa ggccacactg acttcagaca aattttccag cacagcctac 240

atggagctca gcagcctgac ctctgaggac tctgcggtct attactgtac aagacggttg 300

gacgaggctt actggggcca agggactctg gtcactgtct ctgca 345

<210> 136

<211> 115

<212> PRT

<213>Mouse (Mouse)

<400> 136

Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Ser

20 25 30

Val Met His Trp Val Lys Gln Lys Ala Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Gln Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Ser Asp Lys Phe Ser Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Arg Leu Asp Glu Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ala

115

<210> 137

<211> 345

<212> DNA

<213>Mouse (Mouse)

<400> 137

gaggtccagc tgcagcagtc tggacctgag ctggtaaagc ctggggcttc agtgaagatg 60

tcctgcaagg cttctggata cacattcact agctctatta ttcactgggt gaagcagagg 120

cctgggcagg gccttgagtg gattggatat attaatcctt acgatgatgt tactaagtac 180

aatgagaagt tcaaaggcaa ggccacactg acttcagaca aatcctccag cacagcctac 240

atggagctca gcagcctgac ctctgaggac tctgcggtct attactgtgc aagacggtgg 300

gacgagtctt actggggcca agggactctg gtcactgtct ctgca 345

<210> 138

<211> 115

<212> PRT

<213>Mouse (Mouse)

<400> 138

Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Ser

20 25 30

Ile Ile His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Tyr Asp Asp Val Thr Lys Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Ser Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Trp Asp Glu Ser Tyr Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ala

115

<210> 139

<211> 345

<212> DNA

<213>Mouse (Mouse)

<400> 139

gaggtccagc tgcagcagtc tggacctgag ctggtaaagc ctggggcttc agtgaagatg 60

tcctgcaagg cttctggata cactttcact acctctatta tgcactgggt gaaacagaag 120

cctgggcagg gccttgagtg gattggatat attaatcctt acgatgatgt tactaagtac 180

aatgaaaagt tcaaaggcaa ggccacattg acttcagaca aatcctccag cacagcctac 240

atggagctca gcagcctgac ctctgaggac tctgcagtct attactgtgt aagacggtgg 300

gacgaggctt actggggcca agggactctg gtcactgtct ctgca 345

<210> 140

<211> 115

<212> PRT

<213>Mouse (Mouse)

<400> 140

Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Thr Ser

20 25 30

Ile Met His Trp Val Lys Gln Lys Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Tyr Asp Asp Val Thr Lys Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Ser Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Val Arg Arg Trp Asp Glu Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ala

115

<210> 141

<211> 357

<212> DNA

<213>Mouse (Mouse)

<400> 141

gaggtccagc tgcaacaatc tggacctgag ctggtgaagc ctggggcttc agtgaagata 60

tcctgtaagg cttctggata cacgtttact gactactaca tgaactgggt gaggcagagc 120

catggagaga gccttgagtg gattggagat tttaatcata acaatgatgt tattacttac 180

aacccgaagt tcaagggcaa ggtcacattg actgtagaga agtcttccac cacagcctac 240

atggagctcc gcagcctgtc atctgaggac tctgcagtct attactgtgc aagggggcta 300

cgaggctact atgctatgga ctactggggt caaggaacct cagtcaccgt ctcctca 357

<210> 142

<211> 119

<212> PRT

<213>Mouse (Mouse)

<400> 142

Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Tyr Met Asn Trp Val Arg Gln Ser His Gly Glu Ser Leu Glu Trp Ile

35 40 45

Gly Asp Phe Asn His Asn Asn Asp Val Ile Thr Tyr Asn Pro Lys Phe

50 55 60

Lys Gly Lys Val Thr Leu Thr Val Glu Lys Ser Ser Thr Thr Ala Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Ser Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Leu Arg Gly Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Ser Val Thr Val Ser Ser

115

<210> 143

<211> 354

<212> DNA

<213>Mouse (Mouse)

<400> 143

caggttcagc tgcaacagtc tgacgctgag ttggtgaaac ctggagcttc agtgaagata 60

tcctgcaagg tttctggcta caccttcact gaccatacta ttcactggat gaagcagagg 120

cctgaacagg gcctggaatg gattggatat atttatccta gagatggtta tcctaagttc 180

aatgagaagt tcaagggcaa ggccacattg actgcagaca aatcctccag cacagcctac 240

atgcagctca acagcctgac atctgaggac tctgcagtct atttctgtgc aagacggccc 300

ccttactatg ctatggacta ctggggtcaa ggaacctcag tcgccgtctc ctca 354

<210> 144

<211> 118

<212> PRT

<213>Mouse (Mouse)

<400> 144

Gln Val Gln Leu Gln Gln Ser Asp Ala Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Val Ser Gly Tyr Thr Phe Thr Asp His

20 25 30

Thr Ile His Trp Met Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Tyr Pro Arg Asp Gly Tyr Pro Lys Phe Asn Glu Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys

85 90 95

Ala Arg Arg Pro Pro Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Ser Val Ala Val Ser Ser

115

<210> 145

<211> 345

<212> DNA

<213>Mouse (Mouse)

<400> 145

gaggtccaac tgcagcagtc tggacctgag ctggtaaagc ctggggcttc agtgaagatg 60

tcctgcaagg cttctggata cacattcact aggtatctta tgcactgggt gaagcagaag 120

cctgggcagg gccttgagtg gattggttat attaatcctt acaatgatgg tactaattac 180

aatgagaagt tcaaaggcaa ggccacactg acttcagaca aatcctccag cacagcctac 240

atggagctca gcagcctgac ctctgaggac tctgcggtct attactgttc ccttaactgg 300

gactatgctt actggggcca agggactctg gtcactgtct ctgca 345

<210> 146

<211> 115

<212> PRT

<213>Mouse (Mouse)

<400> 146

Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Arg Tyr

20 25 30

Leu Met His Trp Val Lys Gln Lys Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Asn Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Ser Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ser Leu Asn Trp Asp Tyr Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ala

115

<210> 147

<211> 360

<212> DNA

<213>Mouse (Mouse)

<400> 147

gagttccagc tgcagcagtc tggacctgag ctggtgaagc ctggcgcttc agtgaagata 60

tcctgcaagg cttctggtta ctcattcact gactacaaca tgaactgggt gaagcagagc 120

aaaggaaaga gccttgagtg gattggagta attattccta actatggttt tactagctac 180

aatcagaact tcaagggcaa ggccactttg actgtagacc agtcttccag cacagcccac 240

atgcagctca acagtgtgac atctgaggac tctgcagtct attactgtgt aagagatggg 300

ggaatactcc tctggtatct cgatgtctgg ggcacaggga ccacggtcac cgtctcctca 360

<210> 148

<211> 120

<212> PRT

<213>Mouse (Mouse)

<400> 148

Glu Phe Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asp Tyr

20 25 30

Asn Met Asn Trp Val Lys Gln Ser Lys Gly Lys Ser Leu Glu Trp Ile

35 40 45

Gly Val Ile Ile Pro Asn Tyr Gly Phe Thr Ser Tyr Asn Gln Asn Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Val Asp Gln Ser Ser Ser Thr Ala His

65 70 75 80

Met Gln Leu Asn Ser Val Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Val Arg Asp Gly Gly Ile Leu Leu Trp Tyr Leu Asp Val Trp Gly Thr

100 105 110

Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 149

<211> 366

<212> DNA

<213>Mouse (Mouse)

<400> 149

gaggtccagc tgcaacagtc tggacctgag ctggtgaagc ctggggcttc agtgaagata 60

ccctgcaagg cttctggata cacattcact gactacaaca tggactgggt gaagaagagc 120

catggaaaga gccttgagtg gattggagat atcaatcctc acaatggtgg tactatctac 180

aaccagaagt tcaagggcaa ggccacattg actgtagaca agtcctccag cacagcccac 240

atggagctcc gcagcctgac atctgaggac actgcagtct attactgtgc aagaaattac 300

tacggtagta gttacggctg gtacttcgat gtctggggca cagggaccac ggtcaccgtc 360

tcgtca 366

<210> 150

<211> 122

<212> PRT

<213>Mouse (Mouse)

<400> 150

Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Ile Pro Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Asn Met Asp Trp Val Lys Lys Ser His Gly Lys Ser Leu Glu Trp Ile

35 40 45

Gly Asp Ile Asn Pro His Asn Gly Gly Thr Ile Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala His

65 70 75 80

Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asn Tyr Tyr Gly Ser Ser Tyr Gly Trp Tyr Phe Asp Val Trp

100 105 110

Gly Thr Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 151

<211> 360

<212> DNA

<213>Mouse (Mouse)

<400> 151

gaggtccagc tgcagcagtc tggacctgag ctggtgaagc ctggggcttc agtgaagatt 60

tcctgcaggg cttctggtta ctcattcact ggctactaca tgaactgggt gaagcaaagt 120

cctgaaaaga gccttgagtg gattggagag attattccta ccactggtgg tacttcctac 180

aaccagaagt tcaaggccaa ggccacattg actgtagaca aatcctccag cacagcctac 240

atgcagctca agagcctgac atctgaggac tctgcagtct attactgtgc aagagagagc 300

ggtgggttct actggtactt cgatgtctgg ggcacaggga ccacggtcac cgtctcctca 360

<210> 152

<211> 120

<212> PRT

<213>Mouse (Mouse)

<400> 152

Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Ile Ser Cys Arg Ala Ser Gly Tyr Ser Phe Thr Gly Tyr

20 25 30

Tyr Met Asn Trp Val Lys Gln Ser Pro Glu Lys Ser Leu Glu Trp Ile

35 40 45

Gly Glu Ile Ile Pro Thr Thr Gly Gly Thr Ser Tyr Asn Gln Lys Phe

50 55 60

Lys Ala Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Lys Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Ser Gly Gly Phe Tyr Trp Tyr Phe Asp Val Trp Gly Thr

100 105 110

Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 153

<211> 345

<212> DNA

<213>Mouse (Mouse)

<400> 153

gaggtccagc tgcagcagtc tggacctgag ctggtaaagc ctggggcttc agtgaagatg 60

tcctgcaagg cttctggata cacattcact aggtatgtta tgcactgggt gaagcagaag 120

cctgggcagg gccttgagtg gattggatat attaatcctt acaatgatgt tactaagtac 180

aatgagaagt tcaaaggcaa ggccacactg acttcagaca gatcctccag cacagcctac 240

atgaaactca gcagcctgac ctctgaggac tctgcggtct attattgtgc aagaaactgg 300

gacgttcctt actggggcca agggactctg atcactgtct ctgca 345

<210> 154

<211> 115

<212> PRT

<213>Mouse (Mouse)

<400> 154

Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Arg Tyr

20 25 30

Val Met His Trp Val Lys Gln Lys Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Tyr Asn Asp Val Thr Lys Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Ser Asp Arg Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Lys Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asn Trp Asp Val Pro Tyr Trp Gly Gln Gly Thr Leu Ile Thr

100 105 110

Val Ser Ala

115

<210> 155

<211> 345

<212> DNA

<213>Mouse (Mouse)

<400> 155

gaggtccagc tgcagcagtc tggacctgag ctggtaaagc ctggggcttc agtgaagatg 60

tcctgcaagg cttctggata cacattcact aggtatctta tgcactgggt gaagcagaag 120

cctggacagg gccttgagtg gattggatat attaatcctt acaatgatgg tactaagtac 180

aatgagaggt tcaaaggcaa ggccacactg acttcagaca aatcctccag cacagcctac 240

atggagctca gcagcctgac ctctgaggac tctgcggtct attactgtgc aagaaactgg 300

gacgtacctt actggggcca agggactctg gtcactgtct ctgca 345

<210> 156

<211> 115

<212> PRT

<213>Mouse (Mouse)

<400> 156

Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Arg Tyr

20 25 30

Leu Met His Trp Val Lys Gln Lys Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Lys Tyr Asn Glu Arg Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Ser Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asn Trp Asp Val Pro Tyr Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ala

115

<210> 157

<211> 321

<212> DNA

<213>Artificial sequence

<220>

<223>Humanized sequence

<400> 157

gacatccaga tgacccagag cccaagcagc ctgagcgcca gcgtgggcga ccgcgtgacc 60

atcacctgca aggccagccg cgacgtggcc atcgccgtgg cctggtacca gcagaagcca 120

ggcaaggtgc caaagctgct gctgttctgg gccagcaccc gccacaccgg cgtgccagac 180

cgcttcagcg gcagcggcag cggcaccgac ttcaccctga ccatcagcag cctgcagcca 240

gaggacctgg ccgactacta ctgccaccag tacagcagct acccattcac cttcggccag 300

ggcaccaagc tggagatcaa g 321

<210> 158

<211> 107

<212> PRT

<213>Artificial sequence

<220>

<223>Humanized sequence

<400> 158

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Arg Asp Val Ala Ile Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Leu

35 40 45

Phe Trp Ala Ser Thr Arg His Thr Gly Val Pro Asp Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Leu Ala Asp Tyr Tyr Cys His Gln Tyr Ser Ser Tyr Pro Phe

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 159

<211> 321

<212> DNA

<213>Artificial sequence

<220>

<223>Humanized sequence

<400> 159

gacatccaga tgacccagag cccaagcagc ctgagcgcca gcgtgggcga ccgcgtgacc 60

atcacctgca aggccagccg cgacgtggcc atcgccgtgg cctggtacca gcagaagcca 120

ggcaaggtgc caaagctgct gatctactgg gccagcaccc gccacaccgg cgtgccaagc 180

cgcttcagcg gcagcggcag ccgcaccgac ttcaccctga ccatcagcag cctgcagcca 240

gaggacgtgg ccgactactt ctgccaccag tacagcagct acccattcac cttcggcagc 300

ggcaccaagc tggagatcaa g 321

<210> 160

<211> 107

<212> PRT

<213>Artificial sequence

<220>

<223>Humanized sequence

<400> 160

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Arg Asp Val Ala Ile Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile

35 40 45

Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Val Ala Asp Tyr Phe Cys His Gln Tyr Ser Ser Tyr Pro Phe

85 90 95

Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 161

<211> 321

<212> DNA

<213>Artificial sequence

<220>

<223>Humanized sequence

<400> 161

gacatccaga tgacccagag cccaagcagc ctgagcgcca gcgtgggcga ccgcgtgacc 60

atcacctgca aggccagccg cgacgtggcc atcgccgtgg cctggtacca gcagaagcca 120

ggcaaggtgc caaagctgct gatctactgg gccagcaccc gccacaccgg cgtgccaagc 180

cgcttcagcg gcagcggcag ccgcaccgac ttcaccctga ccatcagcag cctgcagcca 240

gaggacgtgg ccgactactt ctgccaccag tacagcagct acccattcac cttcggcagc 300

ggcaccaagc tggagatcaa g 321

<210> 162

<211> 107

<212> PRT

<213>Artificial sequence

<220>

<223>Humanized sequence

<400> 162

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Arg Asp Val Ala Ile Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Leu

35 40 45

Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Val Ala Thr Tyr Tyr Cys His Gln Tyr Ser Ser Tyr Pro Phe

85 90 95

Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 163

<211> 321

<212> DNA

<213>Artificial sequence

<220>

<223>Humanized sequence

<400> 163

gacatccaga tgacccagag cccaagcagc ctgagcgcca gcgtgggcga ccgcgtgacc 60

atcacctgca aggccagccg cgacgtggcc atcgccgtgg cctggtacca gcagaagcca 120

ggcaaggtgc caaagctgct gctgttctgg gccagcaccc gccacaccgg cgtgccagac 180

cgcttcagcg gcagcggcag ccgcaccgac ttcaccctga ccatcagcag cctgcagcca 240

gaggacctgg ccgactacta ctgccaccag tacagcagct acccattcac cttcggcagc 300

ggcaccaagc tggagatcaa g 321

<210> 164

<211> 107

<212> PRT

<213>Artificial sequence

<220>

<223>Humanized sequence

<400> 164

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Arg Asp Val Ala Ile Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Leu

35 40 45

Phe Trp Ala Ser Thr Arg His Thr Gly Val Pro Asp Arg Phe Ser Gly

50 55 60

Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Leu Ala Asp Tyr Tyr Cys His Gln Tyr Ser Ser Tyr Pro Phe

85 90 95

Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 165

<211> 360

<212> DNA

<213>Artificial sequence

<220>

<223>Humanized sequence

<400> 165

caggtgcagc tggtgcagag cggcgccgag gtgaagaagc caggcagcag cgtgaaggtg 60

agctgcaagg ccagcggcta caccttcacc gaccagacca tccactggat gcgccaggcc 120

ccaggccagg gcctggagtg gatcggctac atctacccac gcgacgacag cccaaagtac 180

aacgagaact tcaagggcaa ggtcaccatc accgccgaca agagcaccag caccgcctac 240

atggagctga gcagcctgcg cagcgaggac accgccgtgt actactgcgc catcccagac 300

cgcagcggct acgcctggtt catctactgg ggccagggca ccctggtgac cgtgagcagc 360

<210> 166

<211> 120

<212> PRT

<213>Artificial sequence

<220>

<223>Humanized sequence

<400> 166

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Gln

20 25 30

Thr Ile His Trp Met Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Tyr Pro Arg Asp Asp Ser Pro Lys Tyr Asn Glu Asn Phe

50 55 60

Lys Gly Lys Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ile Pro Asp Arg Ser Gly Tyr Ala Trp Phe Ile Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 167

<211> 360

<212> DNA

<213>Artificial sequence

<220>

<223>Humanized sequence

<400> 167

caggtgcagc tggtgcagag cggcgccgag gtgaagaagc caggcagcag cgtgaaggtg 60

agctgcaagg ccagcggctt caccttcacc gaccagacca tccactgggt gcgccaggcc 120

ccaggccagg gcctggagtg gatgggctac atctacccac gcgacgacag cccaaagtac 180

aacgagaact tcaagggcaa ggtcaccctg accgccgaca agagcaccag caccgcctac 240

atggagctga gcagcctgcg cagcgaggac accgccgtgt actactgcgc catcccagac 300

cgcagcggct acgcctggtt catctactgg ggccagggca ccctggtgac cgtgagcagc 360

<210> 168

<211> 120

<212> PRT

<213>Artificial sequence

<220>

<223>Humanized sequence

<400> 168

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Thr Phe Thr Asp Gln

20 25 30

Thr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Tyr Ile Tyr Pro Arg Asp Asp Ser Pro Lys Tyr Asn Glu Asn Phe

50 55 60

Lys Gly Lys Val Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ile Pro Asp Arg Ser Gly Tyr Ala Trp Phe Ile Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 169

<211> 360

<212> DNA

<213>Artificial sequence

<220>

<223>Humanized sequence

<400> 169

caggtgcagc tggtgcagag cggcgccgag gtgaagaagc caggcagcag cgtgaagacc 60

agctgcaagg ccagcggcgg caccttcacc gaccagacca tccactgggt gcgccagcgc 120

ccaggccagg gcctggagtg gatgggctac atctacccac gcgacgacag cccaaagtac 180

aacgagaact tcaagggccg cgtcaccatc accgccgaca agagcaccag caccgcctac 240

atggagctga gcagcctgcg cagcgaggac accgccgtgt actactgcgc catcccagac 300

cgcagcggct acgcctggtt catctactgg ggccagggca ccctggtgac cgtgagcagc 360

<210> 170

<211> 120

<212> PRT

<213>Artificial sequence

<220>

<223>Humanized sequence

<400> 170

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Thr Ser Cys Lys Ala Ser Gly Gly Thr Phe Thr Asp Gln

20 25 30

Thr Ile His Trp Val Arg Gln Arg Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Tyr Ile Tyr Pro Arg Asp Asp Ser Pro Lys Tyr Asn Glu Asn Phe

50 55 60

Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ile Pro Asp Arg Ser Gly Tyr Ala Trp Phe Ile Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 171

<211> 360

<212> DNA

<213>Artificial sequence

<220>

<223>Humanized sequence

<400> 171

caggtgcagc tggtgcagag cggcgccgag gtgaagaagc caggcagcag cgtgaaggtg 60

agctgcaagg ccagcggcgg caccttcacc gaccagacca tccactgggt gcgccaggcc 120

ccaggccagg gcctggagtg gatgggctac atctacccac gcgacgacag cccaaagtac 180

aacgagaatt tcaagggccg cgtcaccctg accgccgaca agagcaccag caccgcctac 240

atggagctga gcagcctgcg cagcgaggac accgccgtgt acttctgcgc ccgcccagac 300

cgcagcggct acgcctggtt catctactgg ggccagggca ccctggtgac cgtgagcagc 360

<210> 172

<211> 120

<212> PRT

<213>Artificial sequence

<220>

<223>Humanized sequence

<400> 172

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Thr Asp Gln

20 25 30

Thr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Tyr Ile Tyr Pro Arg Asp Asp Ser Pro Lys Tyr Asn Glu Asn Phe

50 55 60

Lys Gly Arg Val Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Phe Cys

85 90 95

Ala Arg Pro Asp Arg Ser Gly Tyr Ala Trp Phe Ile Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 173

<211> 642

<212> DNA

<213>Artificial sequence

<220>

<223>Humanized sequence

<400> 173

gacatccaga tgacccagag cccaagcagc ctgagcgcca gcgtgggcga ccgcgtgacc 60

atcacctgca aggccagccg cgacgtggcc atcgccgtgg cctggtacca gcagaagcca 120

ggcaaggtgc caaagctgct gatctactgg gccagcaccc gccacaccgg cgtgccaagc 180

cgcttcagcg gcagcggcag ccgcaccgac ttcaccctga ccatcagcag cctgcagcca 240

gaggacgtgg ccgactactt ctgccaccag tacagcagct acccattcac cttcggcagc 300

ggcaccaagc tggagatcaa gcgtactgtg gctgcaccat ctgtcttcat cttcccgcca 360

tctgatgagc agttgaaatc tggaactgcc tctgttgtgt gcctgctgaa taacttctat 420

cccagagagg ccaaagtaca gtggaaggtg gataacgccc tccaatcggg taactcccag 480

gagagtgtca cagagcagga cagcaaggac agcacctaca gcctcagcag caccctgacg 540

ctgagcaaag cagactacga gaaacacaaa gtctacgcct gcgaagtcac ccatcagggc 600

ctgagctcgc ccgtcacaaa gagcttcaac aggggagagt gt 642

<210> 174

<211> 214

<212> PRT

<213>Artificial sequence

<220>

<223>Humanized sequence

<400> 174

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Arg Asp Val Ala Ile Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile

35 40 45

Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Val Ala Asp Tyr Phe Cys His Gln Tyr Ser Ser Tyr Pro Phe

85 90 95

Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 175

<211> 1347

<212> DNA

<213>Artificial sequence

<220>

<223>Humanized sequence

<400> 175

caggtgcagc tggtgcagag cggcgccgag gtgaagaagc caggcagcag cgtgaaggtg 60

agctgcaagg ccagcggcta caccttcacc gaccagacca tccactggat gcgccaggcc 120

ccaggccagg gcctggagtg gatcggctac atctacccac gcgacgacag cccaaagtac 180

aacgagaact tcaagggcaa ggtcaccatc accgccgaca agagcaccag caccgcctac 240

atggagctga gcagcctgcg cagcgaggac accgccgtgt actactgcgc catcccagac 300

cgcagcggct acgcctggtt catctactgg ggccagggca ccctggtgac cgtgagcagc 360

gcctccacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 420

ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 480

tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 540

ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc 600

tacatctgca acgtgaatca caagcccagc aacaccaagg tcgacaagag agttgagccc 660

aaatcttgtg acaaaactca cacatgccca ccgtgcccag caccagaagc tgctggggga 720

ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 780

gaggtcacat gcgtcgtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 840

tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac 900

agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 960

gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 1020

aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggaggag 1080

atgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 1140

gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 1200

ctggactccg acggctcctt cttcctctac agcaagctca ccgtggacaa gagcaggtgg 1260

cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 1320

cagaagagcc tctccctgtc tccgggt 1347

<210> 176

<211> 449

<212> PRT

<213>Artificial sequence

<220>

<223>Humanized sequence

<400> 176

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Gln

20 25 30

Thr Ile His Trp Met Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Tyr Pro Arg Asp Asp Ser Pro Lys Tyr Asn Glu Asn Phe

50 55 60

Lys Gly Lys Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ile Pro Asp Arg Ser Gly Tyr Ala Trp Phe Ile Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly

<210> 177

<211> 1347

<212> DNA

<213>Artificial sequence

<220>

<223>Humanized sequence

<400> 177

caggtgcagc tggtgcagag cggcgccgag gtgaagaagc caggcagcag cgtgaaggtg 60

agctgcaagg ccagcggctt caccttcacc gaccagacca tccactgggt gcgccaggcc 120

ccaggccagg gcctggagtg gatgggctac atctacccac gcgacgacag cccaaagtac 180

aacgagaact tcaagggcaa ggtcaccctg accgccgaca agagcaccag caccgcctac 240

atggagctga gcagcctgcg cagcgaggac accgccgtgt actactgcgc catcccagac 300

cgcagcggct acgcctggtt catctactgg ggccagggca ccctggtgac cgtgagcagc 360

gcctccacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 420

ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 480

tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 540

ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc 600

tacatctgca acgtgaatca caagcccagc aacaccaagg tcgacaagag agttgagccc 660

aaatcttgtg acaaaactca cacatgccca ccgtgcccag caccagaagc tgctggggga 720

ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 780

gaggtcacat gcgtcgtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 840

tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac 900

agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 960

gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 1020

aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggaggag 1080

atgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 1140

gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 1200

ctggactccg acggctcctt cttcctctac agcaagctca ccgtggacaa gagcaggtgg 1260

cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 1320

cagaagagcc tctccctgtc tccgggt 1347

<210> 178

<211> 449

<212> PRT

<213>Artificial sequence

<220>

<223>Humanized sequence

<400> 178

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Thr Phe Thr Asp Gln

20 25 30

Thr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Tyr Ile Tyr Pro Arg Asp Asp Ser Pro Lys Tyr Asn Glu Asn Phe

50 55 60

Lys Gly Lys Val Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ile Pro Asp Arg Ser Gly Tyr Ala Trp Phe Ile Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly

<210> 179

<211> 642

<212> DNA

<213>Artificial sequence

<220>

<223>Humanized sequence

<400> 179

gacatccaga tgacccagag cccaagcagc ctgagcgcca gcgtgggcga ccgcgtgacc 60

atcacctgca aggccagccg cgacgtggcc atcgccgtgg cctggtacca gcagaagcca 120

ggcaaggtgc caaagctgct gctgttctgg gccagcaccc gccacaccgg cgtgccagac 180

cgcttcagcg gcagcggcag cggcaccgac ttcaccctga ccatcagcag cctgcagcca 240

gaggacctgg ccgactacta ctgccaccag tacagcagct acccattcac cttcggccag 300

ggcaccaagc tggagatcaa gcgtactgtg gctgcaccat ctgtcttcat cttcccgcca 360

tctgatgagc agttgaaatc tggaactgcc tctgttgtgt gcctgctgaa taacttctat 420

cccagagagg ccaaagtaca gtggaaggtg gataacgccc tccaatcggg taactcccag 480

gagagtgtca cagagcagga cagcaaggac agcacctaca gcctcagcag caccctgacg 540

ctgagcaaag cagactacga gaaacacaaa gtctacgcct gcgaagtcac ccatcagggc 600

ctgagctcgc ccgtcacaaa gagcttcaac aggggagagt gt 642

<210> 180

<211> 214

<212> PRT

<213>Artificial sequence

<220>

<223>Humanized sequence

<400> 180

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Arg Asp Val Ala Ile Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Leu

35 40 45

Phe Trp Ala Ser Thr Arg His Thr Gly Val Pro Asp Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Leu Ala Asp Tyr Tyr Cys His Gln Tyr Ser Ser Tyr Pro Phe

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 181

<211> 189

<212> PRT

<213>Homo sapiens (Homo sapiens)

<400> 181

Met Leu Gly Ser Arg Ala Val Met Leu Leu Leu Leu Leu Pro Trp Thr

1 5 10 15

Ala Gln Gly Arg Ala Val Pro Gly Gly Ser Ser Pro Ala Trp Thr Gln

20 25 30

Cys Gln Gln Leu Ser Gln Lys Leu Cys Thr Leu Ala Trp Ser Ala His

35 40 45

Pro Leu Val Gly His Met Asp Leu Arg Glu Glu Gly Asp Glu Glu Thr

50 55 60

Thr Asn Asp Val Pro His Ile Gln Cys Gly Asp Gly Cys Asp Pro Gln

65 70 75 80

Gly Leu Arg Asp Asn Ser Gln Phe Cys Leu Gln Arg Ile His Gln Gly

85 90 95

Leu Ile Phe Tyr Glu Lys Leu Leu Gly Ser Asp Ile Phe Thr Gly Glu

100 105 110

Pro Ser Leu Leu Pro Asp Ser Pro Val Gly Gln Leu His Ala Ser Leu

115 120 125

Leu Gly Leu Ser Gln Leu Leu Gln Pro Glu Gly His His Trp Glu Thr

130 135 140

Gln Gln Ile Pro Ser Leu Ser Pro Ser Gln Pro Trp Gln Arg Leu Leu

145 150 155 160

Leu Arg Phe Lys Ile Leu Arg Ser Leu Gln Ala Phe Val Ala Val Ala

165 170 175

Ala Arg Val Phe Ala His Gly Ala Ala Thr Leu Ser Pro

180 185

Claims

1. humanization anti-il-23 p 19 antibodies or its antigen-binding fragment, the wherein antibody or its antigen-binding fragment include：

A) light chain variable district, the light chain variable district include amino acid sequence SEQ ID NO:19(CDR1-L)；Amino acid sequence SEQ ID NO:20(CDR2-L)；And amino acid sequence SEQ ID NO:21(CDR3-L)；And

B) weight chain variable district, the weight chain variable district include amino acid sequence SEQ ID NO:66 or 67 (CDR1-H)；Amino acid sequence Arrange SEQ ID NO:64(CDR2-H)；And amino acid sequence SEQ ID NO:65 (CDR3-H),

Wherein the antibody or its antigen-binding fragment have the K for being less than 40pM to IL-23_D。

2. the humanization anti-il-23 p 19 antibodies or its antigen-binding fragment of claim 1, the wherein antibody or its antigen binding Fragment includes：

B) weight chain variable district, the weight chain variable district include amino acid sequence SEQ ID NO:66(CDR1-H)；Amino acid sequence SEQ ID NO:64(CDR2-H)；And amino acid sequence SEQ ID NO:65(CDR3-H).

3. the humanization anti-il-23 p 19 antibodies or its antigen-binding fragment of claim 1, the wherein antibody or its antigen binding Fragment includes：

B) weight chain variable district, the weight chain variable district include amino acid sequence SEQ ID NO:67(CDR1-H)；Amino acid sequence SEQ ID NO:64(CDR2-H)；And amino acid sequence SEQ ID NO:65(CDR3-H).

4. any one of claim 1-3 humanization anti-il-23 p 19 antibodies, wherein the antibody is monoclonal antibody.

5. any one of claim 1-4 antibody or antigen-binding fragment, it is used to use in medicine.

6. the antibody or antigen-binding fragment of claim 5, the wherein use are treatment inflammatory disease, autoimmune disease, exhaled Desorption system disease, dysbolism or cancer.

7. the antibody or antigen-binding fragment of claim 6, wherein this is used closes for treatment psoriasis, inflammatory bowel disease, psoriasis Save inflammation, multiple sclerosis, rheumatoid arthritis, Crohn's disease, ulcerative colitis or ankylosing spondylitis.

8. the antibody or antigen-binding fragment of claim 6, it is treatment asthma or COPD (chronic obstructive pulmonaries that wherein this, which is used, Disease).

9. pharmaceutical composition, it includes any one of claim 1-3 antibody or antigen-binding fragment and pharmaceutically acceptable Supporting agent.